Metallothioneins (MT), small molecular weight metal binding proteins are known to play an important protective role against heavy metal toxicity, either as antioxidants or pre-oxidants. However, the mode of metabolic fate of MTs in various metal complexes is not clearly understood. This study was carried out to better understand the mode of selective turnover rate of various form of MT in complexes with different metals. The degradation of in vitro translated mouse 35S-cysteine-MT was examined in lysosomal or cytosolic fractions from mouse liver by gel electrophoresis and autoradiography. Overnight incubations of MT showed extensive proteolysis in the lysosomal fraction but not in cytosolic fractions. However, Cu2+-MT was found to be stable under the same experimental condition. In contrast, Zn did not interfere with MT degradation. These results suggest that lysosomes are chiefly responsible for MT removal and appears to be selective on the metals involved in the MT complex. In vitro, translated, radiolabeled MT provides a suitable substrate for investigating the characteristics of MT degradation.
Animal ; Copper/*metabolism/pharmacology ; Ions ; Liver/drug effects/*metabolism ; Lysosomes/metabolism ; Metallothionein/drug effects/*metabolism ; Mice ; Sulfur Radioisotopes ; Support, Non-U.S. Gov't ; Zinc/*metabolism/pharmacology

PURPOSE: Isosulfan blue has been traditionally used as a tracer to map the lymphatic system during identification of the sentinel lymph node (SLN). However, this vital dye is difficult to obtain in Korea. Radioisotopes such as technetiumlabeled sulfur colloid or albumin colloid are also expensive and complex to use. The purpose of this study is to evaluate usefulness of a sentinel lymph node biopsy (SLNB) using methylene blue dye in breast cancer patients. METHODS: We evaluated the sentinel node mapping experience using methylene blue dye from July 2003 to January 2007. Fifty-eight patients with clinical T1-T2 breast cancer without palpable axillary lymph nodes were enrolled. All SLNs were submitted for intraoperative frozen section and hematoxyline and eosin (H & E) stain analysis. For the negative SLNs, serial sections of each SLN specimen were examined by permanent H & E staining and by immunohistochemical techniques (IHC) using cytokeratin. Regardless of the results of a frozen section for the SLNs, a backup level II or III axillary lymph node dissections (ALND) was performed. RESULTS: Of the 58 patients that underwent a SLNB using methylene blue dye, an SLN was identified in 56 patients (96.6%), and metastatic SLNs were detected in 14 cases. Axillary lymph node metastasis found in 18 out of 58 patients. Thus, the false negative rate for a SLNB was 22.2% (4/18). Two patients had a micrometastasis (pN1mi) and two patients had clusters of isolated tumor cells (pN0[i+]) that were identified in the SLNs by IHC with the additional use of cytoketatin. The sensitivity, specificity, and accuracy of the SLNBs were 77.8%, 100%, and 92.9%, respectively. The false negative rate improved with the accumulation of experience for performing a SLNB (12.5% vs 30.0%). The sensitivity, specificity, positive predictive value, and accuracy of preoperative ultrasonography (USG) for an axillary lymph node metastsis were 50.0%, 95.5%, 81.8% and 81.0% respectively. CONCLUSION: Based on our initial experience, methylene blue dye is safe, inexpensive, and a readily available tracer for the SLN mapping, and it could be an effective alternative to the use of isosulfan blue dye for accurately identifying SLNs in early breast caner patients. We expected that the findings of preoperative USG could serve as useful adjuncts to a SLNB.
Biopsy* ; Breast Neoplasms* ; Breast* ; Colloids ; Eosine Yellowish-(YS) ; Frozen Sections ; Hematoxylin ; Humans ; Keratins ; Korea ; Lymph Node Excision ; Lymph Nodes* ; Lymphatic System ; Methylene Blue* ; Neoplasm Metastasis ; Neoplasm Micrometastasis ; Radioisotopes ; Sensitivity and Specificity ; Sentinel Lymph Node Biopsy ; Sulfur ; Ultrasonography

The presence of heparan sulfate proteoglycan (HSPG) in anionic sites in the lamina rara interna of glomerular basement membrane suggests that the proteoglycan may be deposited by the glomerular endothelial cells (GEndo). We have previously demonstrated that bovine GEndo in vitro synthesize perlecan, a species of glomerular basement membrane HSPG. In this study we examined whether high glucose medium regulates the GEndo metabolism of glycopeptides including perlecan. Metabolic labeling of glycoconjugates with 35S-SO4, sequential ion exchange and Sepharose CL-4B chromatography of labeled glycoconjugates, and northern analysis were performed. Incubation of GEndo for 8 to 14 weeks (but not for 1-2 weeks) in medium containing 30 mM glucose resulted in nearly 50% reduction in the synthesis of cell layer and medium 35SO4-labeled low anionic glycoproteins and proteoglycans, including that of basement membrane HSPG (Kav 0.42) compared to GEndo grown in 5 mM glucose medium; no changes in anionic charge density or hydrodynamic size of proteoglycans were noted. Northern analysis demonstrated that the mRNA abundance of perlecan was reduced by 47% in cells incubated with 30 mM glucose. Our data suggest that high glucose medium reduces the GEndo synthesis of perlecan by regulating its gene expression. Reduced synthesis of perlecan by GEndo may contribute to proteinuria seen in diabetic nephropathy.
Animals ; Basement Membrane/drug effects/metabolism ; Cattle ; Cells, Cultured ; Diabetic Nephropathies/metabolism ; Endothelial Cells/cytology/*drug effects/*metabolism ; Gene Expression/drug effects ; Glucose/*pharmacology ; Heparan Sulfate Proteoglycan/genetics/*metabolism ; Kidney Glomerulus/*cytology ; Sulfur Radioisotopes/diagnostic use ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, Non-P.H.S. ; Support, U.S. Gov't, P.H.S.