Galectin-7 as a marker of cholesteatoma residue and its detection during surgery by an immunofluorescent method--a preliminary study.

OBJECTIVES: To visualize the distribution of galectin-7 in middle ear cholesteatomas using an immunofluorescent method and to establish whether galectin-7 can be used as a marker of cholesteatoma residue at the time of operation. METHODS: Middle ear cholesteatomas were obtained at surgery from 30 patients. Samples were frozen and preserved in a freezer until histological study. After serial sectioning with a cryostat, 2 of the specimens were processed with primary antibody and Zenon rabbit immunoglobulin G labeling kits. After sufficient reaction time, the samples were observed using a confocal laser microscope. In the remaining 28 specimens, the cholesteatoma was treated as 1 block and stained with the same solution. It was then observed using a fluorescent stereomicroscope. RESULTS: Confocal microscopic analyses showed that galectin-7 was distributed in the cholesteatoma matrix. Because this area strongly stained green, it was easily recognized using a confocal laser microscope. In the stereomicroscopic study using the 1-block specimen in which the cholesteatoma was processed together with the surrounding granulation and mucosal tissue, only the matrix and overlying debris was yellow-green in response to excitation by light; the surrounding granulation and mucosal tissues did not respond in 7 specimens. In the remaining 21 specimens, the whole sample was composed of cholesteatoma and responded well to excitation by light. These findings suggest that galectin-7 might be a useful marker of cholesteatoma residue that can be visualized using this immunofluorescent method. CONCLUSION: Because residual cholesteatoma matrix is considered to be one of the main causes of cholesteatoma recurrence, staining with galectin-7 at the time of operation would be a promising way to facilitate complete removal of the residue.

Up-regulation of 42 kDa tubulin alpha-6 chain fragment in well-differentiated hepatocellular carcinoma tissues from patients infected with hepatitis C virus.

We performed proteomic differential display analysis of hepatitis C virus-associated 21 human hepatocellular carcinoma (HCV-HCC) tissues by using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). One of the numerous spots which was located next to three spots of glutamine synthetase showed stronger intensity in well-differentiated HCC tissues compared to non-cancerous tissues. Samples from 6 out of 21 patients showed up-regulation of this spot compared to non-cancerous tissues. After in-gel digestion, MALDITOF/MS identified the spot as tubulin alpha-6 chain. Two-dimensional immunoblot analysis confirmed that this spot was indeed tubulin alpha, and this spot was stronger in cancerous tissues than in noncancerous tissues. These results suggest that tubulin alpha-6 chain is one of the candidates for biomarkers for well-differentiated HCV-associated HCC.

Molecular characterization of Ciona sperm outer arm dynein reveals multiple components related to outer arm docking complex protein 2.

Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Ciona intestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2.

Proteomic profiling of proteins decreased in hepatocellular carcinoma from patients infected with hepatitis C virus.

Hepatocellular carcinoma (HCC) is a major cause of death in Japan. It has been suggested that hepatitis C virus (HCV) plays an important role in hepatocarcinogenesis, because of high incidence among the patients. To understand the mechanism of hepatocarcinogenesis after HCV infection, we performed a comparative study on the protein profiles between tumorous and nontumorous specimens from the patients infected with HCV by means of two-dimensional electrophoresis. Eleven spots were decreased in HCC tissues from over 50% of the patients. Eight proteins out of 11 spots were identified using peptide mass fingerprinting with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. These proteins were liver type aldolase, tropomyosin beta-chain, ketohexokinase, enoyl-CoA hydratase, albumin, smoothelin, ferritin light chain, and arginase 1. The intensity of enoyl-CoA hydratase, tropomyosin beta-chain, ketohexokinase, liver type aldolase, and arginase 1 was significantly different (p < 0.05). The decrease of 8 proteins was characteristic in HCC. We will discuss the implication of these proteins for the loss of function of hepatocytes and for the possibility of carcinogenesis of HCV-related HCC.

Identification of 36 kDa phosphoprotein in fibrous sheath of hamster spermatozoa.

In our previous studies (Fujinoki et al., 2001, 2003), we reported that two types of 36 kDa proteins, designated 36K-A protein and 36K-B protein, obtained from hamster sperm flagella, are associated with motility activation and phosphorylated in a cAMP-dependent manner at serine residues. In the present experiments, we focused on the hamster (Mesocricetus auratus) 36K-A protein, which was analyzed by peptide mass finger printing and amino acid sequencing. The results suggest that 36K-A protein is a pyruvate dehydrogenase E1 component beta subunit lacking the N-terminal 30 amino acids. Moreover, our results suggest that 36 K-A protein is localized in the fibrous sheath of the principal piece of hamster spermatazoa.

Proteomic profiling of heat shock protein 70 family members as biomarkers for hepatitis C virus-related hepatocellular carcinoma.

To identify proteins linked to the pathogenesis of hepatocellular carcinoma (HCC) associated with hepatitis C virus (HCV), we profiled protein expression levels in samples of HCC. To identify essential proteins, ten samples of HCV-related HCC were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. These experiments revealed increased levels of nine proteins in cancerous tissues compared to levels in corresponding noncancerous liver tissues. We focused on four members of the heat shock protein 70 family: 78 kDa glucose-regulated protein (GRP78), heat shock cognate 71 kDa protein (HSC70), 75 kDa glucose-regulated protein (GRP75), and heat shock 70 kDa protein 1 (HSP70.1). These results were confirmed by immunoblot analysis. In an additional 11 samples, the same expression patterns of these four proteins were observed. In total, 21 samples showed statistically significant up-regulation of GRP78, GRP75 and HSP70.1 in cancerous tissues. HSC70 showed a tendency toward overexpression. There has been no report describing overexpression of these four proteins simultaneously in HBV-related HCC as well as nonviral HCC. Our results suggest that these four proteins play important roles in the pathogenesis of HCV-related HCC and could be molecular targets for diagnosis and treatment of this disease.

Identification of the 58-kDa phosphoprotein associated with motility initiation of hamster spermatozoa.

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that the 58-kDa protein obtained from hamster sperm flagella was phosphorylated at serine residues in association with the start of motility. In the present experiments, we identified and localized the 58-kDa protein. The 58-kDa protein was assumed to exist in the acrosomal region domain of the sperm head and the whole sperm flagellum. In particular, a large amount of 58-kDa protein was localized in the equatorial segment of the acrosomal region domain of the sperm head and the middle piece of the sperm flagellum. In the next step, the 58-kDa protein was identified by peptide mass finger printing and LC-MS/MS analysis. The results suggested that the 58-kDa protein was ATP synthase H(+) transporting F1 beta, which is one of the mitochondrial components. Therefore, it is likely that the 58-kDa protein is associated with ATP production in the mitochondrial sheath in the middle piece of the sperm flagellum, and H(+) transport in the sperm head and the sperm flagellum except for the middle piece, since ATP synthase also acts as an H(+) pump.

Identification of 36-kDa flagellar phosphoproteins associated with hamster sperm motility.

In our previous paper [M. Fujinoki et al. (2001) BIOMED: Res. 22, 45-58], we reported that two types of 36-kDa protein, which were designated as 36K-A protein and 36K-B protein, obtained from hamster sperm flagella were phosphorylated at serine residues associated with the regulation of motility activation. In the present experiments, it was suggested that these two types of 36-kDa protein were phosphorylated in a cAMP-dependent manner associated with motility activation of hamster spermatozoa. Because the 36K-B protein was the most intensely phosphorylated in a cAMP-dependent manner, attempts were made to further characterize it. The 36K-B protein was assumed to be localized in the middle piece. The localization of the 36K-B protein was the same as that of the 36-kDa protein reported in our previous paper [Y. Si et al. (1999) Mol. Reprod. Dev. 52, 328-334]. In order to identify the 36K-B protein, it was analyzed by peptide mass finger printing and amino acid sequencing. The results suggested that the 36K-B protein was a pyruvate dehydrogenase E1 component beta subunit and a component of the mitochondrial sheath of the middle piece.