ABSTRACT
We previously reported that pyrrolidine dithiocarbamate blocked nuclear factor-kappaB (NF-kappaB) activation and attenuated interstitial inflammation and tubulointerstitial fibrosis in the rat obstructive nephropathy. Since pyrrolidine dithiocarbamate is an anti-oxidant and possesses additional biological properties, present experiment was conducted to clarify further the role of NF-kappaB in the development of tubulointerstitial fibrosis in obstructed kidney using a proteasome inhibitor that blocks NF-kappaB through stabilizing IkappaB, an endogenous inhibitor of NF-kappaB. At 5 days following unilateral ureteral obstruction (UUO) in rats, obstructed kidney exhibited tubulointerstitial fibrosis that was associated with macrophage infiltration. UUO decreased renal cortical IkappaB protein contents with concomitant increases in NF-kappaB DNA-binding activity and gene expression of monocyte chemoattractant protein-1. Administration of PSI, N-benzyloxy-carbonyl-Ile-Glu (O-t-Bu)-Ala-leucinal, a proteasome inhibitor, (3 mg/kg/day, s.c., b.i.d) to UUO rats inhibited proteasome activity and attenuated the changes in IkappaB content, NF-kappaB activity and MCP-1 mRNA expression observed in UUO rats. PSI also decreased macrophage influx and attenuated the development of fibrosis. Furthermore, up-regulated gene expression of pro-fibrogenic molecules observed in the obstructed kidney was attenuated by PSI. These results further support the notion that NF-kappaB plays an important role in the development of renal fibrosis in the obstructive nephropathy.
Subject(s)
Kidney/pathology , Multienzyme Complexes/antagonists & inhibitors , NF-kappa B/physiology , Animals , Blotting, Northern , Blotting, Western , Cell Nucleus/metabolism , Chemokine CCL2/biosynthesis , Chemokine CCL2/metabolism , Cysteine Endopeptidases , Cytoplasm/metabolism , DNA/chemistry , DNA/metabolism , Fibrosis , I-kappa B Proteins/pharmacology , Kidney/drug effects , Kidney Cortex/pathology , Macrophages/metabolism , Monocytes/metabolism , NF-kappa B/metabolism , Proteasome Endopeptidase Complex , Protein Binding , RNA, Messenger/metabolism , Rats , Time Factors , Up-RegulationABSTRACT
We developed a competitive enzyme-linked immunosorbent assay (ELISA) to detect prion protein contained in materials derived from cattle, aiming at establishing a method to detect abnormal prion protein (PrPSc) in food products. Rabbit polyclonal antibodies were raised against bovine prion peptides. Using these antibodies, we have established a competitive ELISA that is capable of detecting recombinant bovine prion protein (rBoPrP) in the range of 12 to 1,200 ng and we used it to determine prion protein contents in bovine cerebral cortex. This assay system was evaluated by spiking food products with various amounts of rBoPrP. The determination gave 2-fold higher values in minced meat homogenates and lower values in large intestine homogenates than the values expected from the spiked amounts. This assay provides a simple determination method of spiked rBoPrP, and therefore is expected to be useful for investigating sample pretreatment methods.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Food Analysis/methods , Prions/analysis , Animals , Brain Chemistry , Cattle , Meat Products/analysis , Prions/immunology , Recombinant Proteins , TunaABSTRACT
Human glioblastoma cell line T98G produced a cellular form of prion protein (PrP(C)), and we confirmed expression of PrP mRNA by RT-PCR. Immunoblot analysis of whole cell lysate revealed one major (35 kDa) and two faint bands (31, 25 kDa) that reacted with monoclonal anti-human PrP antibody 3F4. Cells treated with tunicamycin produced only a 25 kDa band, representing a deglycosylated form of PrP. Similarly, peptide: N-glycosidase F treatment of whole cell lysate altered the Asn-linked form to the deglycosylated form. When T98G cells were cultured for a longer period, the amount of PrP(C) per cell increased on Day 4 to 16 in a time-dependent manner. When the cells were cultured at high cell-density, the cells on Day 4 produced the same amount of PrP(C) as those on Day 16 of the usual culture. Moreover, in a serum-free medium, cells cultured at a low cell-density produced the same amount of PrP(C) as those cultured at the high cell-density. These results demonstrate that PrP(C) production in T98G cells was dependent on the phase of the cell cycle, probably the G1 phase.
