Simian virus 40-transformed metallothionein null cells showed increased sensitivity to cadmium but not to zinc, copper, mercury or nickel

Primary cultured embryonic cells derived from mice with disrupted metallothionein (MT) I and II genes and from control mice were transformed with a plasmid encoding the simian virus 40 (SV40) large T antigen. The resulting MT-/- and MT+/+ cell strains showed similar cell morphology, cell cycle and no significant differences in glutathione levels or in the activities of glutathione-related enzymes and antioxidant enzymes. The MT-/- cells were more sensitive to Cd than MT+/+ cells, though no increase in the sensitivity to Zn, Cu, Hg or Ni were observed in MT-/- cells. MT+/+ cells accumulated more Cd than MT-/- cells but showed less lesion, suggesting the role of MT induced by Cd in MT+/+ cells as a scavenger of toxic Cd ion. These results suggest a dominant protective role of MT against Cd compared with other metals. SV40-transformed MT-/- cells seem to be a useful tool for the investigation of cellular function of MT.     

3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factor II receptor from the early endosome to the TGN

3-Methyladenine (3-MA), a well-known inhibitor of autophagic sequestration, can also prevent class III phosphatidylinositide (PI) 3-kinase activity, which is required for many processes in endosomal membrane trafficking. Although much is known about the effects of other PI 3-kinase inhibitors, such as wortmannin and LY294002, on endosomal membrane trafficking, little is known about those of 3-MA. Here we show that the treatment of cells with 3-MA results in a specific redistribution of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR300) from the trans-Golgi network (TGN) to early/recycling endosomal compartments containing internalized transferrin. Importantly, in contrast to wortmannin and LY294002, 3-MA did not cause the enlargement of late endosomal/lysosomal compartments. The results suggest that the effect of 3-MA is restricted to the retrieval of MPR300 from early/recycling endosomes.     

Insights into the roles of cathepsins in antigen processing and presentation revealed by specific inhibitors

Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papain-like cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, alpha-fodrin, in Sjogren's syndrome and suppresses the Th-1 response and autoantibody production.     

Endothelin-1 Binding to Endothelin Receptors in the Rat Anterior Pituitary Gland: Interaction in the Recognition of Endothelin-1 Between ETA and ETB Receptors

1. ¹²⁵I-Endothelin (ET)-1 binding to the rat anterior pituitary gland was saturable and single, with a K _d of 71 pM and a B _max of 120 fmol/mg.2. When 1.0 M BQ-123 (ET_A antagonist) was added to the incubation buffer, the binding parameters were 8.3 pM and 8.0 fmol/mg, whereas 10 nM sarafotoxin S6c (ET_Bagonist) exerted little change in these binding parameters (K _d,72pM;B _max, 110 fmol/mg).3. ET_B receptor-related compounds such as sarafotoxin S6c, ET-3, IRL1620, and BQ-788 competitively inhibited ¹²⁵I-ET-1 binding, only when 1.0 M BQ-123 was present in the incubation buffer.4. Thus, the ET_B receptor is capable of binding ET-1 when the ET_A receptor is being occupied by BQ-123. A collaboration mechanism between the ET_A and the ET_B receptor may function in the recognition of ET-1, a typical bivalent ligand.     

Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.