Estudos in silico da proteína AlkB e prospecção de bactérias alcanotróficas em solos da bacia petrolífera potiguar

Some microorganisms from virgin ecosystems are able to use petroleum it as source of carbon and energy. The knowledge of microbial biodiversity can help to reveal new metabolic systems for utilization alkanes with biotechnological importance. The aim of this study is: i) Accomplish an in silico study of the AlkB protein aimed to understand the probable mechanism involved on selectivity of alkanes in Gram positive and Gram negative bactéria. ii) prospect and analyze the response of the microbial alkanotrophics communities in soil and mangrove sediments of BPP RN and soil of Atlantic forest in the Horto Dois Irmãos Reserve area/PE using the molecular biomarker, gene alkB; with the PCR and PCR-DGGE approach

Modulação da expressão da proteína XPA em resposta ao tratamento com azul de metileno fotossensibilizado

Reactive oxygen species (ROS) are produced by aerobic metabolism and react with biomolecules, such as lipids, proteins and DNA. In high concentration, they lead to oxidative stress. Among ROS, singlet oxygen (1O2) is one of the main ROS involved in oxidative stress and is one of the most reactive forms of molecular oxygen. The exposure of some dyes, such as methylene blue (MB) to light (MB+VL), is able to generate 1O2 and it is the principle involved in photodynamic therapy (PDT). 1O2 e other ROS have caused toxic and carcinogenic effects and have been associated with ageing, neurodegenerative diseases and cancer. Oxidative DNA damage is mainly repaired by base excision repair (BER) pathway. However, recent studies have observed the involvement of nucleotide excision repair (NER) factors in the repair of this type of injury. One of these factors is the Xeroderma Pigmentosum Complementation Group A (XPA) protein, which acts with other proteins in DNA damage recognition and in the recruitment of other repair factors. Moreover, oxidative agents such as 1O2 can induce gene expression. In this context, this study aimed at evaluating the response of XPA-deficient cells after treatment with photosensitized MB. For this purpose, we analyzed the cell viability and occurrence of oxidative DNA damage in cells lines proficient and deficient in XPA after treatment with MB+VL, and evaluated the expression of this enzyme in proficient and complemented cells. Our results indicate an increased resistance to treatment of complemented cells and a higher level of oxidative damage in the deficient cell lines. Furthermore, the treatment was able to modulate the XPA expression up to 24 hours later. These results indicate a direct evidence for the involvement of NER enzymes in the repair of oxidative damage. Besides, a better understanding of the effects of PDT on the induction of gene expression could be provided

Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins

Caracterização funcional de dois cDNAs homólogos à ciclofilina e à proteína reprimida por auxina (ARP) identificados em bibliotecas subtrativas a para floração em tomateiro

Flowering is a fundamental process in the life cycle for plant. This process is marked by vegetative to reproductive apical meristem conversion, due to interactions between several factors, both internal and external to plant. Therefore, eight subtractive libraries were constructed using apical meristem induced or not induced for two contrasting species: Solanum lycopersicum cv. Micro-Tom and Solanum pimpinellifolium. Several cDNAs were identified and among these, were selected two cDNAs: one homologous cDNA to cyclophilin (LeCYP1) and the other to Auxin repressed protein (ARP). It has observed that LeCYP1 and ARP genes are important in the developmental process to plants. In silico analysis, were used several databases with the exclusion criterion E-value <1.0x10-15. As a result, conservation was observed for proteins analyzed by means of multiple alignments and the presence of functional domains. Then, overexpression cassettes were constructed for the ARP cDNA in sense and antisense orientations. For this step, it was used the CaMV35S promoter. The cDNA orientation (sense or antisense) in relation to the promoter was determined by restriction enzymes and sequencing. Then, this cassette was transferred to binary vector pZP211 and these cassettes were transferred into Agrobacterium tumefaciens LBA4404. S. lycopersicum cv. Micro-Tom (MT) and MT-Rg1 plants were transformed. In addition, seedlings were subjected to hormone treatments using a synthetic auxin (- naphthalene acetic acid) and cyclosporin A (cyclophilin inhibitor) treatments and it was found that the hormone treatment there were changes in development of lateral roots pattern, probably related to decreases in auxin signaling caused by reduction of LeCYP1 in MT-dgt plants while cyclosporin A treatments, there was a slight delay in flowering in cv. MT plants. Furthermore, assay with real-time PCR (RT-qPCR) were done for expression level analysis from LeCYP1 and ARP in order to functionally characterize these sequences in tomato plants.

Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition

Análise metagenômica da microbiota de ambientes aquáticos do estado do Rio Grande do Norte - Brasil

The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundiaí-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundiaí river

Análise das mutações C282Y e H63D no gene da proteína HFE em pacientes com hiperferritinemia

Hereditary Hemochromatosis (HH) is a genetic disease caused by high iron absorption and deposition in several organs. This accumulation results in clinical disturbances such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders and skin darkening. The H63D and C282Y mutations are well defined in the hemochromatosis etiology. The aim of this paper was that of identifying the H63D and C282Y genetical mutations in the hemochromatosis gene and the frequency assessment of these mutations in the HFE protein gene in patients with hyperferritin which are sent to the DNA Center laboratory in Natal, state of Rio Grande do Norte. This paper also evaluates the HH H63D and C282Y gene mutations genotype correlation with the serum ferritin concentration, glucose, alanine aminotransferasis, aspartato aminotransferasis, gama glutamil transferasis and with the clinical complications and also the interrelation with life habits including alcoholism and iron overload. The biochemical dosages and molecule analyses are done respectively by the enzymatic method and PCR with enzymatic restriction. Out of the 183 patients investigated, 51,4% showed no mutation and 48,6% showed some type of mutation: 5,0% were C282Y heterozygous mutation; 1,1%, C282Y homozygous mutation; 31%, H63D heterozygous mutation; 8,7%, H63D homozygous mutation; and 3,3%, heterozygous for the mutation in both genes. As to gender, we observed a greater percentage of cases with molecular alteration in men in relation to women in the two evaluated mutations. The individuals with negative results showed clinical and lab signs which indicate hemochromatosis that other genes could be involved in the iron metabolism. Due to the high prevalence of hemochromatosis and taking into account that hemochromatosis is considered a public health matter, its gravity being preventable and the loss treatment toxicity, the early genetic diagnosis is indicated, especially in patients with high ferritin, and this way it avoids serious clinical manifestations and increases patients' life expectation. Our findings show the importance of doing such genetic studies in individuals suspected of hereditary hemochromatosis due to the high incidence of such a hereditary disease in our region

Estudo das interações polifenol-proteína e das reações de escurecimento não-enzimático para o processamento de cajuína

The cashew, a fruit from Brazilian Northeast is used to produce juice due to its flavor and vitamin C richness. However, its acceptance is limited due to its astringency. Cajuína is a derivate product appreciated by its characteristic flavor, freshness and lack of astringency, due to tannin removal. Cajuína is a light yellow beverage made from clarified cashew juice and sterilized after bottling. It differs from the integral and concentrated juice by the clarification and thermal treatment steps. Many problems such as haze and excessive browning could appear if these steps are not controlled. The objective of this work was divided into two stages with the aim to supply process information in order to obtain a good quality product with uniform characteristics (sensory and nutritional). Polyphenol-protein interaction was studied at the clarification step, which is an empirical process, to provide values on the amount of clarifying solution (gelatin) that must be added to achieve a complete juice clarification. Clarification essays were performed with juice dilutions of 1:2 and 1:10 and the effect of metabissulfite and tannic acid addition was evaluated. It was not possible to establish a clarification point. Metabissulfite did not influenced the clarification process however tannic acid addition displaced the clarification point, showing the difficulty visual monitoring of the process. Thermal treatment of clarified juice was studied at 88, 100, 111 e 121 °C. To evaluate the non-enzymatic browning, vitamin C, 5-hidroximetilfurfural (5-HMF) and sugar variation were correlated with color parameters (reflectance spectra, color difference and CIELAB). Kinetic models were obtained for reflectance spectra, ascorbic acid and 5-HMF. It was observed that 5-HMF introduction followed a first order kinetic rate at the beginning of the thermal treatment and a zero order kinetic at later process stages. An inverse correlation was observed between absorbance at 420 nm and ascorbic acid degradation, which indicates that ascorbic acid might be the principal factor on cajuína non-enzymatic browning. Constant sugar concentration showed that this parameter did not contribute directly to the nonenzymatic browning. Optimization techniques showed showed that to obtain a high vitamin C and a low 5-HMF content, the process must be done at 120 ºC. With the water-bath thermal treatment, the 90 °C temperature promoted a lower ascorbic acid degradation at the expense of a higher 5-HMF level

Avaliação dos níveis de proteína C-reativa ultra-sensível em pacientes com periodontite crônica severa generalizada e sem periodontite

INTRODUCTION: The high sensitivity C-reactive protein (hsCRP) constitutes an inflammatory mediator used as predictor of cardiovascular risk that comes being researched as indicative relation factor between cardiovascular and periodontal diseases. PROPOSITION: To compare serumals levels of C-reactive protein between patients with and without generalized severe chronic periodontitis. METHODOLOGY: A seccional study was realized using a sample with 62 patients, being 31 participants carriers of periodontal diseases (Group I) and 31 without periodontal diseases (Group II), grouped to the pairs by age and sex. As inclusion criterio were selected patients with diagnosis of generalized severe chronic periodontitis, being preculeds, individuals which presented systemic disease, recent infection history, historical of CVA or stroke, smokers, pregnants and lactants. The research consisted of two stages, a clinc and other biochemist. The clinical stage is constituted of periodontal examination and the biochemist stage, of the peripheral blood collection for determination hsCRP levels and a hemogram to inquire any panel which could suggest infectious and/or inflammatory process. RESULTS: Periodontal disease group presented a average of 0,36mg/dL, while the group without disease presented 0,17 mg/dL, do not existing significant difference statistically between the averages (p = 0,061). The cardiovascular risk for the group I was classified high for 27,6% of participants and low for 72,4% of them. In the group II, 6,45% presented high risk e 93,5% low risk, being this significant relation statistically gotten for Fisher s Test (p = 0,042) presenting OR = 5,33; IC = 95% (1,02 27,4). The independets variables reseacred do not presented significant association statistically with the levels of hsCRP. CONCLUSION: The study indicated that despite of carriers patients of periodontal diseases do not present differents serumals levels of hsCRP from the other group, the periodontal disease was considered as risk factor for hsCRP plasmatic levels elevation

Influência do tratamento periodontal sobre os níveis da proteína c-reativa ultrassensível em pacientes com insuficiência renal crônica em fase de prétransplante

The present study evaluated the influence of non-surgical periodontal treatment on the levels of C- reactive protein (hsCRP) in patients with chronic renal failure (CRF) in pretransplant. We conducted a controlled and randomized trial to evaluate the periodontal condition and plasma concentrations of hsCRP, albumin and transferrin in 56 dialysis patients divided into two groups: experimental and control. The study was conducted at the dental clinic of Family and Community Health s Unit (USFC), located in Onofre Lopes University Hospital (HUOL), Federal University of Rio Grande do Norte (UFRN), from December 2010 to November 2011. Severe periodontitis was the type of periodontal disease more common, affecting 78.6% of patients. Periodontal conditions, evaluated through the means of probing depth, clinical attachment level, bleeding index and plaque index, proved to be uniform for both groups at the initial examination. There were no differences in levels of inflammatory markers between the two groups. The analysis of the concentrations of hsCRP allowed classifying study participants as at high risk of developing cardiovascular disease. After completion of periodontal treatment in the experimental group, there was a statistically significant reduction of the mean of all periodontal parameters assessed; however this improvement of periodontal health was not accompanied by changes in the levels of hsCRP, albumin and transferrin in the evaluation time. Given this, the periodontal treatment did not promote the reduction of systemic inflammatory burden and risk of cardiovascular complications in patients with CRF

Estudo da detecção do DNA do papiloma vírus humano (HPV) e da expressão imuno-histoquímica de proteína do ciclo celular no carcinoma epidermóide oral

Oral squamous cell carcinoma (OSCC) is the most common malignancy in oral cavity and human papillomavirus (HPV) may have an important role in its development. The aim of this experiment was to investigate the HPV DNA and viral types in 90 cases of OSCC. Moreover, a comparative analysis between the cases of OSSC with and without HPV DNA was performed by using cell cycle markers p21 and pRb in order to detect a possible correlation of these proteins and HPV infection. DNA was extracted from paraffin embedded tissue and amplified by PCR (polymerase chain reaction) with primers PCO3+ e PCO4+ for a fragment of human β-globin gene. After this procedure, PCR for HPV DNA detection was realized using a pair of generic primers GP5+ e GP6+. Immunohistochemical study was performed by streptoavidin-biotin technique and antibodies against p21 and pRb proteins were employed. Eighty-eight cases were positive for human β-globin gene and HPV DNA was found in 26 (29.5%) of then. It could not be detected significant correlation between HPV and age, sex and anatomical sites of the lesion. The most prevalent viral type was HPV 18 (80.8%). Regarding the immunohistochemical analysis, it was detected significant association between HPV presence and pRb immunoexpression (p=0,044), nevertheless, the same was not observed in relation to p21 protein (p =0,416). It can be concluded that the low detection of HPV DNA in OSCC by the present experiment suggests a possible role of the virus in the development and progression in just a subset of this disease

Eosinófilos e proteína catiônica eosinofílica na urina: uma nova abordagem para o diagnóstico da inflamação renal no lúpus eritematoso sistêmico

Universidade Estadual do Rio Grande do Norte