Júlio César Da Silva - Academia.edu (original) (raw)

Papers by Júlio César Da Silva

Revista Arvore, 2003

The objective of this study was to determine the working conditions and training level of forest ... more The objective of this study was to determine the working conditions and training level of forest fire volunteers of the Jardim Botânico de Brasília, IBGE Ecological Reserve and Água Limpa Farm, Universidade de Brasília. The study was carried out during the first semester of 2000 on the headquarters of the three brigades located in each Conservation Unit. The methodology consisted in applying questionnaires and interviews to the brigadiers and their coordinators. The data set was analyzed for the three brigades, with the best training level and brigadier satisfaction being found at the IBGE Ecological Reserve, and the worst at the UnB´s Água Limpa Farm. No individual protection equipment was available to the forest fire fighters and the number of forest fire fighting equipments and tools was not sufficient. No road maintenance machines were at the disposal of the brigades. Only the IBGE Ecological Reserve Brigade was supplied with a pipe truck for forest fire fighting. Água Limpa farm Brigade had the greatest participation in educational campaigns at the nearby communities. It was concluded that all the brigades have a good training and skill level and despite the limitation of tools and equipments, they are able to fight small forest fires. The worst problem found was the lack of individual protection during forest fire fighting, what may cause accidents.

Acta Cirurgica Brasileira, 2006

PURPOSE: Study the proliferation rate of jejunum and large intestine crypt epithelial cells, in r... more PURPOSE: Study the proliferation rate of jejunum and large intestine crypt epithelial cells, in rats pinealectomized immediately after borning. METHODS: Twenty-four male Wistar rats were distributed into two groups: Acute group (n=12) and Chronic group (n=12). Six animals of each group were operated for removal of the pineal gland (pinealectomy-PnX), and other six were controls (sham pinealectomy-C). Animals from acute and chronic group were sacrificed 15 and 90 days after the surgery, respectively. RESULTS: In acute group, pinealectomy of new-born rats has not caused significant alteration in cell proliferation (PnX=58,77±1,77 and C=60,88±1,10 in the descending colon/ PnX=31,56±0,45 and C=31,73±0,47 in the proximal jejunum) and in crypt cell population (PnX=24,92±4,82 and C=23,60±2,48 in the descending colon/ PnX=39,92±3,49 and C=44,32±5,56 in the proximal jejunum). However, in chronic group there was an uprising crypt cell production per crypt in the proximal jejunum (PnX=57,54±2,19 and C=47,19±7,3)and in the descending colon (PnX=37,78±2,22 and C=17,92±2,28). CONCLUSION: As the increase of intestinal crypts epithelial cells in chronic group is a carcinogenesis predetermining factor, the understanding of the interaction between pineal gland and this event has great importance.

BMC Structural Biology, 2009

Background: Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates... more Background: Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates, which have been functionally implicated in Calcium homeostasis. However, recent data from mammalian systems indicated that they may be also involved in embryogenesis, tumorigenesis and in the context of the latter especially in angiogenesis. Human STC1 is a 247 amino acids protein with a predicted molecular mass of 27 kDa, but preliminary data suggested its di-or multimerization. The latter in conjunction with alternative splicing and/or post-translational modification gives rise to forms described as STC 50 and "big STC", which molecular weights range from 56 to 135 kDa.

Plant Physiology and Biochemistry, 2009

The aldo-keto reductases (AKRs) are classified as oxidoreductases and are found in organisms from... more The aldo-keto reductases (AKRs) are classified as oxidoreductases and are found in organisms from prokaryotes to eukaryotes. The AKR superfamily consists of more than 120 proteins that are distributed throughout 14 families. Very few plant AKRs have been characterized and their biological functions remain largely unknown. Previous work suggests that AKRs may participate in stress tolerance by detoxifying reactive aldehyde species. In maize endosperm, the presence of an aldose reductase (AR; EC 1.1.1.21) enzyme has also been hypothesized based on the extensive metabolism of sorbitol. This manuscript identifies and characterizes an AKR from maize (Zea mays L.) with features of an AR. The cDNA clone, classified as AKR4C7, was expressed as a recombinant His-tag fusion protein in Escherichia coli. The product was purified by immobilized metal affinity chromatography followed by anion exchange chromatography. Circular dichroism spectrometry and SAXS analysis indicated that the AKR4C7 protein was stable, remained folded throughout the purification process, and formed monomers of a globular shape, with a molecular envelope similar to human AR. Maize AKR4C7 could utilize dl-glyceraldehyde and some pentoses as substrates. Although the maize AKR4C7 was able to convert sorbitol to glucose, the low affinity for this substrate indicated that AKR4C7 was probably a minimal contributor to sorbitol metabolism in maize seeds. Polyclonal antisera raised against AKR4C7 recognized at least three AR-like polypeptides in maize kernels, consistent with the presence of a small gene family. Diverse functions may have evolved for maize AKRs in association with specific physiological requirements of kernel development.

BMC Structural Biology, 2011

Background The NIMA-related kinases (Neks) are widespread among eukaryotes. In mammalians they re... more Background The NIMA-related kinases (Neks) are widespread among eukaryotes. In mammalians they represent an evolutionarily conserved family of 11 serine/threonine kinases, with 40-45% amino acid sequence identity to the Aspergillus nidulans mitotic regulator NIMA within their catalytic domains. Neks have cell cycle-related functions and were recently described as related to pathologies, particularly cancer, consisting in potential chemotherapeutic targets. Human Nek6, -7 and -9 are involved in the control of mitotic spindle formation, acting together in a mitotic kinase cascade, but their mechanism of regulation remain elusive. Results In this study we performed a biophysical and structural characterization of human Nek6 with the aim of obtaining its low resolution and homology models. SAXS experiments showed that hNek6 is a monomer of a mostly globular, though slightly elongated shape. Comparative molecular modeling together with disorder prediction analysis also revealed a flexible disordered N-terminal domain for hNek6, which we found to be important to mediate interactions with diverse partners. SEC-MALS experiments showed that hNek6 conformation is dependent on its activation/phosphorylation status, a higher phosphorylation degree corresponding to a bigger Stokes radius. Circular dichroism spectroscopy confirmed our in silico predictions of secondary structure content and thermal stability shift assays revealed a slightly higher stability of wild-type hNek6 compared to the activation loop mutant hNek6(S206A). Conclusions Our data present the first low resolution 3D structure of hNek6 protein in solution. SAXS, comparative modeling and SEC-MALS analysis revealed that hNek6 is a monomeric kinase of slightly elongated shape and a short unfolded N-terminal domain.

Biopolymers, 2007

We regret any confusion caused by this error.

Biopolymers, 2007

The conformational changes and aggregation process of β-lactoglobulin (β-LG) subjected to gamma i... more The conformational changes and aggregation process of β-lactoglobulin (β-LG) subjected to gamma irradiation are presented. β-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (aw) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1–50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of β-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated β-LG in solid state at different aw and dose level was essentially the same as the nonirradiated β-LG. The scattering data also showed that the irradiation of β-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of β-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS–PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric β-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of β-LG dimers subunits, while the samples irradiated in the solid state were not modified. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 284–294, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Journal of Proteome Research, 2008

The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 mono... more The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1, in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Smallangle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/ 57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein.

BMC Structural Biology, 2010

Background The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified a... more Background The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.

Journal of Chromatography A, 2009

Counter-current chromatography (CCC) was used to isolate chavibetol from the essential oil of lea... more Counter-current chromatography (CCC) was used to isolate chavibetol from the essential oil of leaves of Pimenta pseudocaryophyllus (Gomes) Landrum. Chavibetol was obtained in high purity (98%) and mass recovery (94.4%). Methyleugenol was also isolated. The CCC biphasic solvent system used was composed of hexane:n-butanol:methanol:water (12:4:4:3, v/v/v/v).

Revista Arvore, 2003

The objective of this study was to determine the working conditions and training level of forest ... more The objective of this study was to determine the working conditions and training level of forest fire volunteers of the Jardim Botânico de Brasília, IBGE Ecological Reserve and Água Limpa Farm, Universidade de Brasília. The study was carried out during the first semester of 2000 on the headquarters of the three brigades located in each Conservation Unit. The methodology consisted in applying questionnaires and interviews to the brigadiers and their coordinators. The data set was analyzed for the three brigades, with the best training level and brigadier satisfaction being found at the IBGE Ecological Reserve, and the worst at the UnB´s Água Limpa Farm. No individual protection equipment was available to the forest fire fighters and the number of forest fire fighting equipments and tools was not sufficient. No road maintenance machines were at the disposal of the brigades. Only the IBGE Ecological Reserve Brigade was supplied with a pipe truck for forest fire fighting. Água Limpa farm Brigade had the greatest participation in educational campaigns at the nearby communities. It was concluded that all the brigades have a good training and skill level and despite the limitation of tools and equipments, they are able to fight small forest fires. The worst problem found was the lack of individual protection during forest fire fighting, what may cause accidents.

Acta Cirurgica Brasileira, 2006

PURPOSE: Study the proliferation rate of jejunum and large intestine crypt epithelial cells, in r... more PURPOSE: Study the proliferation rate of jejunum and large intestine crypt epithelial cells, in rats pinealectomized immediately after borning. METHODS: Twenty-four male Wistar rats were distributed into two groups: Acute group (n=12) and Chronic group (n=12). Six animals of each group were operated for removal of the pineal gland (pinealectomy-PnX), and other six were controls (sham pinealectomy-C). Animals from acute and chronic group were sacrificed 15 and 90 days after the surgery, respectively. RESULTS: In acute group, pinealectomy of new-born rats has not caused significant alteration in cell proliferation (PnX=58,77±1,77 and C=60,88±1,10 in the descending colon/ PnX=31,56±0,45 and C=31,73±0,47 in the proximal jejunum) and in crypt cell population (PnX=24,92±4,82 and C=23,60±2,48 in the descending colon/ PnX=39,92±3,49 and C=44,32±5,56 in the proximal jejunum). However, in chronic group there was an uprising crypt cell production per crypt in the proximal jejunum (PnX=57,54±2,19 and C=47,19±7,3)and in the descending colon (PnX=37,78±2,22 and C=17,92±2,28). CONCLUSION: As the increase of intestinal crypts epithelial cells in chronic group is a carcinogenesis predetermining factor, the understanding of the interaction between pineal gland and this event has great importance.

BMC Structural Biology, 2009

Background: Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates... more Background: Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates, which have been functionally implicated in Calcium homeostasis. However, recent data from mammalian systems indicated that they may be also involved in embryogenesis, tumorigenesis and in the context of the latter especially in angiogenesis. Human STC1 is a 247 amino acids protein with a predicted molecular mass of 27 kDa, but preliminary data suggested its di-or multimerization. The latter in conjunction with alternative splicing and/or post-translational modification gives rise to forms described as STC 50 and "big STC", which molecular weights range from 56 to 135 kDa.

Plant Physiology and Biochemistry, 2009

The aldo-keto reductases (AKRs) are classified as oxidoreductases and are found in organisms from... more The aldo-keto reductases (AKRs) are classified as oxidoreductases and are found in organisms from prokaryotes to eukaryotes. The AKR superfamily consists of more than 120 proteins that are distributed throughout 14 families. Very few plant AKRs have been characterized and their biological functions remain largely unknown. Previous work suggests that AKRs may participate in stress tolerance by detoxifying reactive aldehyde species. In maize endosperm, the presence of an aldose reductase (AR; EC 1.1.1.21) enzyme has also been hypothesized based on the extensive metabolism of sorbitol. This manuscript identifies and characterizes an AKR from maize (Zea mays L.) with features of an AR. The cDNA clone, classified as AKR4C7, was expressed as a recombinant His-tag fusion protein in Escherichia coli. The product was purified by immobilized metal affinity chromatography followed by anion exchange chromatography. Circular dichroism spectrometry and SAXS analysis indicated that the AKR4C7 protein was stable, remained folded throughout the purification process, and formed monomers of a globular shape, with a molecular envelope similar to human AR. Maize AKR4C7 could utilize dl-glyceraldehyde and some pentoses as substrates. Although the maize AKR4C7 was able to convert sorbitol to glucose, the low affinity for this substrate indicated that AKR4C7 was probably a minimal contributor to sorbitol metabolism in maize seeds. Polyclonal antisera raised against AKR4C7 recognized at least three AR-like polypeptides in maize kernels, consistent with the presence of a small gene family. Diverse functions may have evolved for maize AKRs in association with specific physiological requirements of kernel development.

BMC Structural Biology, 2011

Background The NIMA-related kinases (Neks) are widespread among eukaryotes. In mammalians they re... more Background The NIMA-related kinases (Neks) are widespread among eukaryotes. In mammalians they represent an evolutionarily conserved family of 11 serine/threonine kinases, with 40-45% amino acid sequence identity to the Aspergillus nidulans mitotic regulator NIMA within their catalytic domains. Neks have cell cycle-related functions and were recently described as related to pathologies, particularly cancer, consisting in potential chemotherapeutic targets. Human Nek6, -7 and -9 are involved in the control of mitotic spindle formation, acting together in a mitotic kinase cascade, but their mechanism of regulation remain elusive. Results In this study we performed a biophysical and structural characterization of human Nek6 with the aim of obtaining its low resolution and homology models. SAXS experiments showed that hNek6 is a monomer of a mostly globular, though slightly elongated shape. Comparative molecular modeling together with disorder prediction analysis also revealed a flexible disordered N-terminal domain for hNek6, which we found to be important to mediate interactions with diverse partners. SEC-MALS experiments showed that hNek6 conformation is dependent on its activation/phosphorylation status, a higher phosphorylation degree corresponding to a bigger Stokes radius. Circular dichroism spectroscopy confirmed our in silico predictions of secondary structure content and thermal stability shift assays revealed a slightly higher stability of wild-type hNek6 compared to the activation loop mutant hNek6(S206A). Conclusions Our data present the first low resolution 3D structure of hNek6 protein in solution. SAXS, comparative modeling and SEC-MALS analysis revealed that hNek6 is a monomeric kinase of slightly elongated shape and a short unfolded N-terminal domain.

Biopolymers, 2007

We regret any confusion caused by this error.

Biopolymers, 2007

The conformational changes and aggregation process of β-lactoglobulin (β-LG) subjected to gamma i... more The conformational changes and aggregation process of β-lactoglobulin (β-LG) subjected to gamma irradiation are presented. β-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (aw) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1–50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of β-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated β-LG in solid state at different aw and dose level was essentially the same as the nonirradiated β-LG. The scattering data also showed that the irradiation of β-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of β-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS–PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric β-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of β-LG dimers subunits, while the samples irradiated in the solid state were not modified. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 284–294, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Journal of Proteome Research, 2008

The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 mono... more The human protein Ki-1/57 was first identified through the cross reactivity of the anti-CD30 monoclonal antibody Ki-1, in Hodgkin lymphoma cells. The expression of Ki-1/57 in diverse cancer cells and its phosphorylation in peripheral blood leukocytes after mitogenic activation suggested its possible role in cell signaling. Ki-1/57 interacts with several other regulatory proteins involved in cellular signaling, transcriptional regulation and RNA metabolism, suggesting it may have pleiotropic functions. In a previous spectroscopic analysis, we observed a low content of secondary structure for Ki-1/57 constructs. Here, Circular dichroism experiments, in vitro RNA binding analysis, and limited proteolysis assays of recombinant Ki-1/57(122-413) and proteolysis assays of endogenous full length protein from human HEK293 cells suggested that Ki-1/57 has characteristics of an intrinsically unstructured protein. Smallangle X-ray scattering (SAXS) experiments were performed with the C-terminal fragment Ki-1/ 57(122-413). These results indicated an elongated shape and a partially unstructured conformation of the molecule in solution, confirming the characteristics of an intrinsically unstructured protein.

BMC Structural Biology, 2010

Background The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified a... more Background The adaptor protein RACK1 (receptor of activated kinase 1) was originally identified as an anchoring protein for protein kinase C. RACK1 is a 36 kDa protein, and is composed of seven WD repeats which mediate its protein-protein interactions. RACK1 is ubiquitously expressed and has been implicated in diverse cellular processes involving: protein translation regulation, neuropathological processes, cellular stress, and tissue development. Results In this study we performed a biophysical analysis of human RACK1 with the aim of obtaining low resolution structural information. Small angle X-ray scattering (SAXS) experiments demonstrated that human RACK1 is globular and monomeric in solution and its low resolution structure is strikingly similar to that of an homology model previously calculated by us and to the crystallographic structure of RACK1 isoform A from Arabidopsis thaliana. Both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation techniques showed that RACK1 is predominantly a monomer of around 37 kDa in solution, but also presents small amounts of oligomeric species. Moreover, hydrodynamic data suggested that RACK1 has a slightly asymmetric shape. The interaction of RACK1 and Ki-1/57 was tested by sedimentation equilibrium. The results suggested that the association between RACK1 and Ki-1/57(122-413) follows a stoichiometry of 1:1. The binding constant (KB) observed for RACK1-Ki-1/57(122-413) interaction was of around (1.5 ± 0.2) × 106 M-1 and resulted in a dissociation constant (KD) of (0.7 ± 0.1) × 10-6 M. Moreover, the fluorescence data also suggests that the interaction may occur in a cooperative fashion. Conclusion Our SAXS and analytical ultracentrifugation experiments indicated that RACK1 is predominantly a monomer in solution. RACK1 and Ki-1/57(122-413) interact strongly under the tested conditions.

Journal of Chromatography A, 2009

Counter-current chromatography (CCC) was used to isolate chavibetol from the essential oil of lea... more Counter-current chromatography (CCC) was used to isolate chavibetol from the essential oil of leaves of Pimenta pseudocaryophyllus (Gomes) Landrum. Chavibetol was obtained in high purity (98%) and mass recovery (94.4%). Methyleugenol was also isolated. The CCC biphasic solvent system used was composed of hexane:n-butanol:methanol:water (12:4:4:3, v/v/v/v).