Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Background\ud Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray.\ud \ud \ud Results\ud The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue.\ud \ud \ud Conclusions\ud The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.FAPESP doctoral fellowship n°. 05/50781-2CTC/CEPID/FAPESP [grant n. 1998/14247-6

Expression of human protein S100A7 (psoriasin), preparation of antibody and application to human larynx squamous cell carcinoma

Abstract\ud \ud Background\ud Up-regulation of S100A7 (Psoriasin), a small calcium-binding protein, is associated with the development of several types of carcinomas, but its function and possibility to serve as a diagnostic or prognostic marker have not been fully defined. In order to prepare antibodies to the protein for immunohistochemical studies we produced the recombinant S100A7 protein in E. coli. mRNA extracted from human tracheal tumor tissue which was amplified by RT-PCR to provide the region coding for the S100A7 gene. The amplified fragment was cloned in the vector pCR2.1-TOPO and sub-cloned in the expression vector pAE. The protein rS100A7 (His-tag) was expressed in E. coli BL21::DE3, purified by affinity chromatography on an Ni-NTA column, recovered in the 2.0 to 3.5 mg/mL range in culture medium, and used to produce a rabbit polyclonal antibody anti-rS100A7 protein. The profile of this polyclonal antibody was evaluated in a tissue microarray.\ud \ud \ud Results\ud The rS100A7 (His-tag) protein was homogeneous by SDS-PAGE and mass spectrometry and was used to produce an anti-recombinant S100A7 (His-tag) rabbit serum (polyclonal antibody anti-rS100A7). The molecular weight of rS100A7 (His-tag) protein determined by linear MALDI-TOF-MS was 12,655.91 Da. The theoretical mass calculated for the nonapeptide attached to the amino terminus is 12,653.26 Da (delta 2.65 Da). Immunostaining with the polyclonal anti-rS100A7 protein generated showed reactivity with little or no background staining in head and neck squamous cell carcinoma cells, detecting S100A7 both in nucleus and cytoplasm. Lower levels of S100A7 were detected in non-neoplastic tissue.\ud \ud \ud Conclusions\ud The polyclonal anti-rS100A7 antibody generated here yielded a good signal-to-noise contrast and should be useful for immunohistochemical detection of S100A7 protein. Its potential use for other epithelial lesions besides human larynx squamous cell carcinoma and non-neoplastic larynx should be explored in future.FAPES

REE Anomalies Changes in Bottom Sediments Applied in the Western Equatorial Atlantic Since the Last Interglacial

We reconstruct paleoredox conditions in the Western Equatorial Atlantic (WEA) over the glacial-interglacial cycle (~130 ka) by using new high-resolution REEs data and their anomalies from a marine sediment core (GL-1248) collected from the equatorial margin off the continental shelf of NE Brazil. This approach aims to improve the understanding of the dynamics of paleoclimatic and sedimentary inputs on the coast of northeastern Brazil. Marine sediments were analyzed via Mass Spectrometry (ICP-MS) after total digestion with HF/HNO3. REEs proxies are a useful tool in understanding the transport and origin of sediments due to their physicochemical properties. Our data showed the Parnaíba River was the main source of REEs content in the western South Atlantic. Fe minerals (Fe-oxyhydroxides) produced via weathering of continental and tropical soils were the principal REE-carrier phase during transportation and ultimate deposition at core site GL-1248. Several regional climatic factors mainly rainfall changes contributed significantly to continental-REEs erosion of sedimentary layers of the Parnaíba Basin, and transport and deposition of the mobilized REEs from the continent to the study site. Furthermore, changes in the negative Ce-anomaly showed low variation along the core indicating a reduction in deep ocean oxygenation during the interglacial relative to the last glacial period. That variation, probably, was associated with glacial-interglacial variations in sea level with the exposure of the continental shelf. The origin of positive Eu anomalies in siliciclastic sediment, also observed in the core, was explained by preferential retention by feldspars such as plagioclases and potassium feldspars mostly from the assimilation of felspar during fractionation crystallization of felsic magma in the Parnaíba basin since the Last Interglacial

REE Anomalies Changes in Bottom Sediments Applied in the Western Equatorial Atlantic Since the Last Interglacial

We reconstruct paleoredox conditions in the Western Equatorial Atlantic (WEA) over the glacial-interglacial cycle (~130 ka) by using new high-resolution REEs data and their anomalies from a marine sediment core (GL-1248) collected from the equatorial margin off the continental shelf of NE Brazil. This approach aims to improve the understanding of the dynamics of paleoclimatic and sedimentary inputs on the coast of northeastern Brazil. Marine sediments were analyzed via Mass Spectrometry (ICP-MS) after total digestion with HF/HNO3. REEs proxies are a useful tool in understanding the transport and origin of sediments due to their physicochemical properties. Our data showed the Parnaíba River was the main source of REEs content in the western South Atlantic. Fe minerals (Fe-oxyhydroxides) produced via weathering of continental and tropical soils were the principal REE-carrier phase during transportation and ultimate deposition at core site GL-1248. Several regional climatic factors mainly rainfall changes contributed significantly to continental-REEs erosion of sedimentary layers of the Parnaíba Basin, and transport and deposition of the mobilized REEs from the continent to the study site. Furthermore, changes in the negative Ce-anomaly showed low variation along the core indicating a reduction in deep ocean oxygenation during the interglacial relative to the last glacial period. That variation, probably, was associated with glacial-interglacial variations in sea level with the exposure of the continental shelf. The origin of positive Eu anomalies in siliciclastic sediment, also observed in the core, was explained by preferential retention by feldspars such as plagioclases and potassium feldspars mostly from the assimilation of felspar during fractionation crystallization of felsic magma in the Parnaíba basin since the Last Interglacial