Purification and identification of monoubiquitin-phosphoglycerate mutase B complex from human colorectal cancer tissues.

Ubiquitin-conjugated proteins in human colorectal cancer tissues were analyzed by the immunoprecipitation with the antibody FK2 against conjugated ubiquitin followed with SDS-PAGE. In these immunoprecipitable proteins, a 38-kDa protein was abundant in the tumor regions but almost absent in the adjacent normal regions in 17/26 patients, thus we attempted to purify it. Using immunoaffinity chromatography with the antibody FK2 followed by gel filtration and SDS-PAGE, approximately 10 pmol of this protein was separated from 34 g of the pooled cancerous tissue and transferred onto a PVDF membrane. The 38-kDa protein was further digested with Achromobacter protease I, resulting in several peptide fragments. Amino acid sequences of these peptides showed complete sequence identity to those derived from either ubiquitin or phosphoglycerate mutase-B, suggesting that the 38-kDa protein is monoubiquitinated phosphoglycerate mutase-B, whose calculated mass is 37,369 Da. Western blot using an antibody against phosphoglycerate mutase-B revealed the presence of the 38-kDa protein in the anti-ubiquitin immunoprecipitates derived from the tumor regions, but not from normal counterparts. In addition, part of non-ubiquitinated phosphoglycerate mutase-B (29 kDa) was also found in the anti-ubiquitin immunoprecipitates, whose levels were higher in the tumor regions than in the adjacent normal regions. These results suggest that monoubiquitination of phosphoglycerate mutase-B as well as formation of a noncovalent complex containing ubiquitin and phosphoglycerate mutase-B increases in colorectal cancer and novel modification of phosphoglycerate mutase-B might have a pathophysiological role.

Effects of recombinant human erythropoietin (rHuEPO) on nutritional status of hemodialysis patients: investigation of direct anabolic effects of rHuEPO.

To investigate whether the nutritional improvement achieved by recombinant human erythropoietin (rHuEPO) treatment is the result of anemia correction with rHuEPO or the direct anabolic effects of rHuEPO per se, nutritional assessment was performed in 2 studies (study I and II) on hemodialysis (HD) patients. Nutritional assessment included blood biochemistry determinations, anthropometric measurements, daily protein intake (DPI) and dialysis efficiency. In study I, 5 HD patients who had not been given rHuEPO and had a hematocrit (Hct) of < or = 25%, were administered rHuEPO at the initial dose of 96.2 U/kgBW. Nutritional assessment of these patients was performed before rHuEPO treatment and every 4 weeks until the 24th week after rHuEPO treatment. In study II, the same nutritional assessment as in study I except for DPI, was performed in 2 groups with the same Hct level and dialysis regimen; an EPO group (n = 8) previously given rHuEPO (88.2 +/- 13.7 U/kgBW, 25.8 +/- 2.5 mos) and a non-EPO group (n = 8) not given rHuEPO. In study I, the mean Hct level was significantly increased 4 weeks after rHuEPO treatment (23.3 +/- 0.6 to 26.9 +/- 0.9%). However, the nutritional parameters and dialysis efficiency were nearly constant over 24 weeks, suggesting either the absence of a short-term direct anabolic effect of rHuEPO or masking of such an effect due to general condition improvement by anemia correction with rHuEPO. In study II, no significant differences in nutritional assessment were confirmed between the groups, suggesting that a long-term direct anabolic effect of rHuEPO may not exist and nutritional improvement may result from correction of anemia with rHuEPO.(ABSTRACT TRUNCATED AT 250 WORDS)