The HIF-1 inhibitor YC-1 decreases reactive astrocyte formation in a rodent ischemia model.

Astrocytes become reactive after central nervous system injury, re-expressing glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and nestin. Hypoxia-inducible transcription factor alpha (HIF-1α) is an important transcription factor for several genes including the VEGF and nestin genes, the expression of which generate reactive astrocytes and cause gliosis after cerebral ischemia. To evaluate the role of HIF-1α in reactive astrocyte formation, we applied the potent HIF-1α inhibitor YC-1 to a focal cerebral ischemia model and analyzed the expression of HIF-1α, VEGF, nestin, and GFAP. Quantitative real-time reverse transcription polymerase chain reaction and western blot analyses demonstrated that the expression of HIF-1α and its downstream genes (VEGF and nestin) were markedly attenuated in the YC-1-treated group versus the control group (HIF-1α, VEGF: p < 0.01; nestin: p < 0.05). GFAP expression was also effectively inhibited in the YC-1-treated group (p < 0.05). Immunohistochemical evaluations showed that GFAP-positive (GFAP+) cells in the YC-1-treated group were sparse in the peri-infarct area, while an immunofluorescence assay revealed that the number of VEGF+/GFAP+ and nestin+/GFAP+ reactive astrocytes were decreased in the YC-1-treated group (p < 0.05). These results demonstrate that HIF-1α suppression decreases the formation of reactive astrocytes and gliosis that occur following focal ischemia.

VE1 immunohistochemical detection of the BRAF V600E mutation in thyroid carcinoma: a review of its usefulness and limitations.

The BRAF V600E mutation is a valuable prognostic factor in thyroid carcinoma despite lingering debate. Successful immunohistochemical (IHC) detection of the BRAF V600E mutation using a VE1 antibody was introduced recently. The objective of this study was to verify the usefulness of IHC detection of the BRAF V600E mutation in thyroid carcinoma using the VE1 antibody. IHC detection of BRAF V600E was performed on various thyroid carcinoma subtypes. IHC results were compared with those obtained from real-time polymerase chain reaction (PCR) detection. Discordant cases were re-examined using a direct sequencing method following nested PCR amplification. The BRAF V600E mutation was detected in 68 % (71/104) of papillary carcinoma cases and 78 % (7/9) of anaplastic carcinoma cases. The mutation was not detected in patients with follicular carcinoma (0/18) or in medullary carcinoma (0/21). The overall sensitivity and specificity of IHC using the VE1 antibody were 100 and 94 %, respectively, suggesting that molecular-based results were indeterminable in four VE1-positive cases. IHC using the VE1 antibody is a highly sensitive and specific method for BRAF V600E mutation detection and may represent a future replacement for DNA-based molecular tests.

Evaluation of Protein Expression in Housekeeping Genes across Multiple Tissues in Rats.

BACKGROUND: Housekeeping genes, which show constant protein expression patterns between different tissue types, are very important in molecular biological studies as an internal control for protein research. METHODS: The protein expression profiles of seven housekeeping genes (HPRT1, PPIA, GYS1, TBP, YWHAZ, GAPDH and ACTB) in various rat tissues (cerebrum, cerebellum, cardiac ventricle and atrium, psoas muscle, femoral muscle, liver, spleen, kidney, and aorta) were analyzed by Western blot and compared by coefficient of variation (CV). RESULTS: HPRT1 was stably expressed (CV≤10%) in six tissues (cerebrum, cerebellum, ventricle, femoral muscle, spleen, and kidney), PPIA was stably expressed in five tissues (cerebrum, cerebellum, ventricle, spleen and kidney), YWHAZ was stably expressed in three tissues (cerebrum, cerebellum, and kidney), and GAPDH was stably expressed in four tissues (cerebrum, ventricle, psoas muscle, and kidney). In comparison, GYS1, TBP, and ACTB were found to have CV values over 10% in all tissues. Of the seven genes examined, four (HPRT1, PPIA, YWHAZ, and GAPDH) were found to be stably expressed across multiple organs, with low CV values (≤10%). CONCLUSIONS: These results will provide fundamental information regarding internal controls for protein expression studies and can be used for analysis of postmortem protein degradation patterns in forensic medicine.

Granular cell tumours of the colorectum: histopathological and immunohistochemical evaluation of 30 cases.

AIMS: Granular cell tumours (GCTs) are uncommon in the gastrointestinal tract, particularly in the colorectum. Herein, we report a series of 30 colorectal GCTs and discuss the properties of colorectal GCTs based on histopathological and immunohistochemical studies. METHODS AND RESULTS: Searching the surgical pathology files identified 30 cases of colorectal GCTs for 2005-2013. A broad panel of antibodies including neural and macrophage markers were used for immunohistochemical evaluation. Colorectal GCTs predominantly involved the right colon and showed increased nuclear atypia including nuclear pleomorphism and nuclear spindling. All 24 cases with mucosal tumour components had infiltrative growth patterns within the mucosa. In all available cases, diffuse strong immunopositivity was observed for S100 and SOX10 of schwannian differentiation markers, as well as for CD68. Other neuronal lineage markers, including CD56, neuron-specific enolase, nestin, and synaptophysin showed consistently high expression rates. The immunohistochemical results are suggestive for a neural origin of GCTs. CONCLUSION: Histopathological and immunohistochemical features of colorectal GCTs were delineated in this large series of 30 colorectal GCTs. Although the incidence of GCTs is relatively low, clinicians and pathologists need to be aware of GCT in the differential diagnosis.