High expression of HSP47 in ulcerative colitis-associated carcinomas: proteomic approach.

BACKGROUND: Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease, known to be associated with a markedly increased risk of colorectal carcinoma development. METHODS: Using proteomic analysis with two-dimensional gel electrophoresis and mass spectrometry, differentially expressed proteins were assessed between UC-associated cancer and sporadic colon cancer cell lines. Western blot and immunostaining were performed for confirming the expression. RESULTS: Heat-shock protein of 47 kDa (HSP47) was identified as one of the proteins expressed more highly in UC-associated cancer cell lines, and an immunohistochemical examination confirmed significantly higher levels of HSP47 in UC-associated colon cancers than in sporadic counterparts, the expression increasing with a progression of neoplastic lesions. Heat-shock protein of 47 kDa was further found to be coexpressed with type I collagen in the cytoplasm, and both HSP47 and type I collagen were released from cultured cells into the culture medium. CONCLUSION: These results suggest that overexpression of HSP47 is a unique characteristic of UC-associated carcinoma related to type I collagen synthesis, with possible clinical applications.

Proteome analysis of prostate cancer.

In this paper, we briefly review cancer proteomics in general, with particular attention to our proteome analyses of prostate cancer. Our efforts include development of new tools and novel approaches to discovering proteins potentially useful as cancer diagnostic and/or prognostic biomarkers or as therapeutic targets. To this end, we analyzed prostate cancer proteomes using two-dimensional gel electrophoresis employing agarose gels for the initial isoelectric focusing step (agarose 2-DE), with mass spectrometry used for protein identification. Agarose 2-DE offers advantages over the more widely used immobilized pH gradient 2-DE for separating high molecular mass proteins (15-500 kDa), thereby increasing its power to detect changes in the cancer's high-molecular mass proteomes.

Detection and identification of proteins related to the hereditary dwarfism of the rdw rat.

Proteins having relations to hereditary dwarfism of the rdw rat (gene symbol: rdw) were searched for in various tissues of the rat with an improved two-dimensional gel electrophoresis technique followed by immunoblotting and microsequencing. Tissues inspected were cerebral cortex, cerebellum, brain trunk, hypothalamus, pituitary, thyroid gland, liver, testis, spleen, and thymus. Only pituitary and thyroid glands among those tissues showed abnormalities in protein contents. GH and PRL contents in the rdw pituitary were much less than in the normal one, which in the former were 1/15 and less than 1/30 times as much as in the latter, respectively, but the abnormalities in the rdw thyroid were far more serious than in the pituitary. At least 18 protein levels in the rdw thyroid were above, and 17 were below the normal. Those identified among the increased proteins were endoplasmin (GRP94), immunoglobulin heavy chain binding protein (BiP/GRP78), and heat shock protein 70 (hsp70), the contents of which respectively were 40 times, 10 times and more than 50 times as much in the rdw thyroid as in the normal tissue. Because BiP and endoplasmin are known to be ER resident proteins, and because all three belong to a chaperone protein family, accumulation of these proteins in the rdw thyroid suggests that protein folding and secreting disorders underlie the hypothyroidism of the rdw rat.

A novel missense mutation (G2320R) in thyroglobulin causes hypothyroidism in rdw rats.

The rdw rat is a hereditary hypothyroid variant initially derived from the Wistar-Imamichi strain. Proteome analysis by two-dimensional gelelectrophoresis showed that molecular chaperones accumulated in the thyroid glands, suggesting retention of abnormal proteins in the endoplasmic reticulum (ER). Anatomical studies indicated that thyroglobulin (Tg) was not secreted into the follicular lumina, but retained in the dilated ER. Sequencing of the entire Tg complementary DNA from the rdw rat revealed a missense mutation (G2320R) in the acetylcholinesterase-like domain at the 2320th amino acid residue. Carbohydrate residues of the G2320R Tg mutant were of the high-mannose ER type, as shown by sensitivity to the treatment with endoglycosidase H. Molecular chaperones, GRP94, GRP78, and calreticulin, were all accumulated in the rdw rat thyroid glands. Computer analysis of protein secondary structure predicted that the mutation would cause extension of the helix where beta-sheet and turns were formed in the normal Tg. Altered folding of Tg might account for the impaired intracellular transport of Tg and activated premature degradation by the same mechanism as in ER storage diseases.

Two types of aggregate in the cerebral cortex of a seizure-sensitive strain of the Mongolian gerbil.

A 70-kDa protein, P70, found mostly in the pyramidal cells of the cerebral cortex of cobalt-induced epileptogenic rats, has been implicated in epileptogenesis. The presence of a P70-like substance was searched for immunohistochemically in the cerebral cortex of MGS/ldr, a seizure-sensitive strain of the Mongolian gerbil (Meriones unguiculatus) that we previously established. Immunoreactive aggregates were observed in the pyramidal neurons of the motor cortex and the primary somatosensory cortex. Analysis using confocal laser scanning microscopy revealed that the aggregates were often colocalized with a second type of aggregate with red autofluorescence at the marginal zone of the cell somata. Both aggregates appeared and increased before the appearance of generalized tonic-clonic convulsion. These may be involved in some change of physiological function of the cerebral cortex but their presence itself is not enough to determine the occurrence of epileptic seizure because the gerbils that showed no such seizure had both aggregates.

Comparison of protein constituents between atria and ventricles from various vertebrates by two-dimensional gel electrophoresis.

1. Protein constituents of cardiac muscles of 23 species were examined by two-dimensional gel electrophoresis in order to find the difference in protein components between atria and ventricles. 2. Protein compositions of atria were similar to those of ventricles, however, differences were found in myosin and some other proteins in most species. 3. A major protein with molecular weights of 12,000-15,000 daltons was distributed only in atria from mammals. 4. The atrioventricular difference suggested that the ventricular tissue was more specialized in mammals than in birds, as compared with the atrial tissue.

The in vitro effects of glycyrrhizin and the derivatives of glycyrrhetinic acid on the activity of cAMP-dependent protein kinase and phosphorylation of cellular polypeptide by the kinase from Ehrlich ascites tumor cells.

The effects of glycyrrhizin (GL) and the derivatives of glycyrrhetinic acid (GA) on the activity of cAMP-dependent protein kinase (A-kinase) and the phosphorylation of cellular polypeptides by the kinase purified from Ehrlich ascites tumor cells had been investigated in vitro. It was found that (i) the derivatives [3 beta-hydroxy-olean-11,13 (18)-diene-30-oic acid Na, olean-9(11), 12 diene-3 beta, 30-diol-3 beta, 30-di-o-hemiphthalate 2Na and olean-12-ene-3 beta, 30-diol 3 beta, 30-di-o-phosphate 2Na] of GA inhibited the activity of A-kinase at the concentrations higher than 25 microM; (ii), at 10 microM, these derivatives and native GL stimulated the activity of the kinase significantly; and (iii) the inhibitory and stimulatory effects of some GA derivatives were clearly correlated with their chemical structures. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis and two dimensional gel electrophoresis followed by autoradiography detected several acidic polypeptides, including polypeptides with approximate molecular weights of 35,000 (pI 4.3), 27,000 (pI 4.5) and 18,000-21,000 (pI 4.5), phosphorylated by A-kinase, to be functioning as mediators in response to these drugs. This observation suggests that the GL-induced inhibition of phosphorylation of these cellular polypeptides by A-kinase may be physiologically implicated in the biochemical mechanisms involved in the anti-inflammatory effect of the drug.

The stimulatory and inhibitory effects of glycyrrhizin and a glycyrrhetinic acid derivative on phosphorylation of lipocortin I by A-kinase in vitro.

The effects of GL and oGA on the activity (phosphorylation of lipocortin I) by A-kinase were investigated in vitro. It was found that (i) phosphorylation of the 35-36 kDa polypeptide by A-kinase was inhibited selectively when the partially purified kinase fraction from EAT cells was incubated with [gamma-32P]ATP in the presence of 60 microM oGA; (ii) the marked polypeptide was identified as a lipocortin I; and (iii) the activity of the kinase was stimulated about 4-fold by 5 microM oGA, but it was inhibited at doses above 25 microM. The drug-induced inhibition of phosphorylation of lipocortin I by A-kinase may be implicated in the anti-inflammatory effect of the drugs.

Detection and identification of subcutaneous adipose tissue protein related to obesity in New Zealand obese mouse.

New Zealand obese (NZO) mouse, a genetic model of obesity, shows hyperphagia, hyperinsulinemia and leptin resistance. We analyzed subcutaneous adipose tissue proteins in NZO mice with a two-dimensional gel electrophoresis technique followed by protein sequence analysis. NZO mice showed hyperinsulinemia and hyperleptinemia. Abdominal subcutaneous adipose tissue was inspected in NZO and C57BL/6J lean mice. Two-dimensional gel electrophoresis detected 4 spots which were obviously reduced in NZO mice. Those spots were p26, p19, p18 and p15. Internal sequences of the p26 and p15 protein were homologous with those of carbonic anhydrase III, p19 was cytochrome b5, p18 was superoxide dismutase. Serum arachidonic acid level in NZO mice was lower by 80% of C57BL/6J mice. The present study demonstrated the reduction of several enzymes related to lipid metabolism in NZO mice. These data raises the hypothesis that the supposed changes of membrane fluidity caused by altered membrane lipid content may involve central leptin resistance of this model of obesity.

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins.

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.

Purification and characterization of a 100 kDa glycyrrhizin (GL)-binding protein (gp100) as an effective phosphate acceptor for CK-II and the effect of GL on the phosphorylation of gp100 by CK-II in vitro.

By means of successive GL-affinity and Mono S column chromatographies (HPLC), a 100 kDa GL-binding protein (gp100) was purified from the partially purified CK-II fraction of EAT cells as an effective phosphate acceptor for CK-II. It was found that (i) gp100 (pI 9.0) is copurified with CK-II, Hsp-90 and p34 or p70; (ii) gp100 cross-reacts with anti-human GR; (iii) phosphorylation of gp100 by CK-II is significantly stimulated by 1 microM GL or 0.3 microM oGA; and (iv) GL as well as DEX inhibit it at doses above 3 microM. Data are provided to suggest that the GL-induced selective inhibition of the CK-II catalyzed phosphorylation of gp100 may be involved in the anti-inflammatory effects of GL.