Endocrine diseases including hypothyroidism and diabetes show abnormalities in many different organs and tissues. To detect and identify of proteins related to the diseases, we have searched for in various tissues of animal models. (1) The rdw rat with hereditary dwarfism and hypothyroidism: Pituitary, pancreas, adrenal, thyroid and submandibular glands among 12 tissues inspected showed abnormalities in protein contents. Abnormalities in the rdw thyroid gland were far more serious than in the other tissues. Decrease of thyroglobulin contents and accumulation of ER molecular chaperone proteins (BiP and GRP94) in the rdw thyroid suggest that thyroglobulin folding and secreting disorder underlie the hypothyroidism of the rdw rat.
(2) The ob and db mice with hereditary diabetes: Liver, pancreas, spleen, atrium, small intestine, visceral adipose tissue and subcutaneous white adipose tissue (SWAT) among 17 tissues inspected showed abnormalities in protein contents. In particular, abnormalities in the liver and the two adipose tissues both in the ob/ob and db/db mice were more serious than in the other tissues. At least 10 and 24 protein levels in the ob/ob and db/db liver, respectively, were above, and 39 and 36, respectively, were below the normal. Such liver abnormalities in these obese mice suggest that obesity is an important factor affecting liver metabolism.

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Evaluation of secretory proteins from various human cancer cells using the functional assays combined with electrophoresis is an efficient approach to not only characterization and comparison of cell lines but also obtaining novel secretory proteins. Conditioned media obtained from human cell lines were subjected to zymography, reverse-zymography, cell attachment assay and cell migration assay, and revealed to contain several novel proteins such as proteinase, proteinase-inhibitor and attachment factors.

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Protein undergoes a variety of modifications under oxidative stress. They include modification by aldehydes originated from lipid peroxidation and oxidative modification of protein thiol. These reactions represent post-translational proteins modifications, and proteomic approach for identification of target molecules and characterization of modification mechanisms is plausible.

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In these days proteome analyses are performed in the field of apoptosis. There are two methods to identify the proteins related to apoptosis. The first is to identify the proteins whose expression change after adding the stimuli which induce apoptosis in cells. The second is to compare 2DE pattern of intracellular proteins from two cell lines, one is sensitive against the inducer to apoptosis, and the other is resistant against this. We used these two methods and identified some proteins which may be related to apoptosis.
For the first, heat shock (45°C, 30min) induced the over expression of stathmin and thymosin β-4 in human T cell line Jurkat, and decrease of eukaryotic initiation factor 5A in human pancreatic cancer cell line MIA PaCa-2.
For the second, we established TNF-α-resistant cell line cKDH-8/11 and sensitive cell line KDH-8/YK from rat hepatoma cell line KDH-8. From differential display using 2-DE, we detected spot 1 which expressed in only KDH-8/YK, and spot 2 whose expression is increased in cKDH-8/11. The spot 2 was identified as phosphatidylethanolamine-binding protein (PEBP).

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Most of proteins are modified post-translationally to function in a normal fashion. The incompleteness of post-translational modification can cause various diseases. The yeast 26S proteasome is a large protease complex that catalyzes the degradation of ubiquitin-tagged proteins. This complex is composed of two subcomplexes, a catalytic core particle (20S proteasome) and a pair of 19S regulatory particles. There are 28 subunits in the 20S proteasome and at least 17 subunits in the 19S regulatory particle. These subunits are post-translationally modified with functional groups such as phospho, N-acetyl, N-myristoyl groups. The phosphorylated subunits of the 26S proteasome have been identified by two-dimensional electrophoresis and mass spectrometry, and three phosphorylation sites of alpha-7 subunit of the 20S proteasome by mass spectrometry. The N-acetylated subunits also have been identified by two-dimensional electrophoresis. These phosphorylation and N-acetylation of the 26S proteasome were found to affect the proteolytic activity of the 20S proteasome and ATPase activity of the 19S regulatory particle.

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We constructed a large-scale preparative gel electrophoresis device equipped with a 10-cm in diameter gel support column and used it with an electroosmotic medium pump system (EOMPS). Operation of the constructed apparatus was examined and resolution of the protein bands was determined by separation of the marker protein mixture (6.4-198kDa) using SDS-PAGE (Laemmli buffer system). In addition, loading capacity was tested by fractionation of 1-2g of a bovine serum albumin (BSA) Cohn V fraction using native-PAGE, resulted in a reasonable separating profile. The advantages of the present apparatus were considered to be its good resolving power combined with simplicity, despite the large scale, and a high loading capacity. An EOMPS enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a large scale, and the present apparatus is able to purify nearly all types of protein using SDS-PAGE as well as acidic proteins with native-PAGE.

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Fabrication of plastic microchannel system was perfomed by the cutting method. The channel was the size of width 200μm×depth 150μm×length 20mm. The electrophoretic system was isoelectric focusing and applied for the separation of protein mixture. Even in this system, although the fabrication of the channel surface is not refined, the very reproducible patterns were observed. One of the reason for this remarkable result is due to the precise introduction method of samples. The electropherograms were obtained within 3min. and showed excellent separation, which may suggest the practical applications of this system for the high throughput analysis. The recoding of the separating patterns was performed by the digital camera, or with magnifying lens system or with a microscope. And the densitometry was done by image processing method.

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The WHO Hyperlipoproteinemia phenotype was classified automatically by fractionations of cholesterol and TG using agarose gel electrophoresis with differential staining. Using this technique, clinical significance of samples, heretofore unclassifiable due to abnormal fractions, was examined.
The abnormal LDL was TG-rich LDL results in peak TG concentrations consistent with LDL migration. HDL and VLDL fractions are hardly affected. This abnormal band appears with advanced liver function degradation and was classified into 3 types: Type A, a narrow LDL band, Type B, a wide LDL band, and Type C, minimal HDL and VLDL bands. Type B appears associated with malignant tumors.
Lp-X is a Cholesterol rich band and appears electrophoretically on the catholic side of LDL. The presence of Lp-X is associated with jaundice and advanced cholestasis. Lp-Y resembles Lp-X relative to mobility and pathology, but contains Cholesterol and TG. Poor prognosis is anticipated with the presence of these abnormal LDL, Lp-X and Lp-Y fractions.
Slow αHDL bands appear in the patient with slight bile stagnation and is accompanied with increased Apo E with positive prognosis.
IDL, (VLDL remnant), is the TG-rich band appearing between VLDL and LDL. Lp(a) appears between VLDL and LDL, but is distinguishable from IDL due to the“sharp-banding” and its Cholesterol-rich content.
The simultaneous analysis of Cholesterol and TG fractions can satisfactorily interpret the WHO Hyperlipoproteinemia phenotype with their elevated Cholesterol and TG containing fractions. This test is useful in assessing pathology associated with metabolic lipoprotein disorders.

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