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Biomedical subjects

Masaki Kato

Publications and source records attributed to Masaki Kato.

12 recordsLinked to original sources
PubMed2003

STAM proteins bind ubiquitinated proteins on the early endosome via the VHS domain and ubiquitin-interacting motif.

Conjugation with ubiquitin acts as a sorting signal for proteins in the endocytic and biosynthetic pathways at the endosome. Signal-transducing adaptor molecule (STAM) proteins, STAM1 and STAM2, are associated with hepatocyte growth factor-regulated substrate (Hrs) but their function remains unknown. Herein, we show that STAM proteins bind ubiquitin and ubiquitinated proteins and that the tandemly located VHS (Vps27/Hrs/STAM) domain and ubiquitin-interacting motif serve as the binding site(s). STAM proteins colocalize with Hrs on the early endosome. Overexpression of STAM proteins, but not their mutants lacking the ubiquitin-binding activity, causes the accumulation of ubiquitinated proteins and ligand-activated epidermal growth factor receptor on the early endosome. These results suggest that through interaction with ubiquitinated cargo proteins on the early endosome via the VHS domain and ubiquitin-interacting motif, STAM proteins participate in the sorting of cargo proteins for trafficking to the lysosome.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Gene expression profiling reveals the mechanism and pathophysiology of mouse liver regeneration.

Comprehensive analysis of the changes in gene expression during liver regeneration was carried out by using an in-house microarray composed of 2,304 distinct mouse liver cDNA clones. Mice were subjected to partial two-thirds hepatectomy, and changes in mRNA levels were monitored up to 48 h. Of the 2,304 genes analyzed, 496 genes showed expression levels measurable at all time points after the partial hepatectomy. 317 genes were up- or down-regulated 2-fold or more at least at one time point during liver regeneration and were classified into eight clusters based on their expression patterns. With a more stringent cut-off value of +/-2 S.D., 68 genes were listed and were classified into five clusters. In these two analyses with different clustering criteria, functionally categorized genes showed similar cluster distributions. Genes involved in protein synthesis and posttranslational processing were significantly enriched in the cluster characterized by rapid gene activation and subsequent persistence. This suggests the importance of modulating the efficiency of protein supply and/or altering the composition of protein population from the early phase of hepatocyte proliferation. Genes for two major liver functions, i.e. plasma protein secretion and intermediate metabolism were enriched in distinct clusters exhibiting the features of gradual gene activation and sustained repression, respectively. Therefore, these genes are differentially regulated during the regeneration, possibly leading to changes in the flow of amino acids and energy from enzyme proteins to plasma proteins in their synthesis. Thus, clustering analysis of expression patterns of functionally classified genes gave insights into mechanism and pathophysiology of liver regeneration.

Animals↗
PubMed2003

Two-peaked synchronization in day/night expression rhythms of the fibrinogen gene cluster in the mouse liver.

Genes expressed with day/night rhythms in the mouse liver were searched for by microarray analysis using an in-house array harboring mouse liver cDNAs. The rhythmic expression with a single peak and trough level was confirmed by RNA blot analysis for 3beta-Hsd and Gabarapl1 genes exhibiting a peak in the light phase and Spot14, Hspa8, Hspa5, and Hsp84-1 genes showing a peak in the dark phase. On the other hand, mRNA levels for all of the three fibrinogen subunits, Aalpha, Bbeta and gamma, exhibited two peaks each in the light and dark phases in a synchronized manner. This two-peaked rhythmic pattern of fibrinogen genes as well as the single peak-trough pattern of other genes was diminished or almost completely lost in the liver of Clock mutant mice, suggesting that the two-peaked expression is also under the control of oscillation-generating genes. In constant darkness, the first peak of the expression rhythm of fibrinogen genes was almost intact, but the second peak disappeared. Therefore, although the first peak in the subjective day is a component of the innate circadian rhythm, the second peak seems to require light stimuli. Fasting in constant darkness caused shifts of time phases of the circadian rhythms. Protein levels of the fibrinogen subunits in whole blood also exhibited circadian rhythms. In the mouse and human loci of the fibrinogen gene cluster, a number of sequence elements resembling circadian transcription factor-binding sites were found. The fibrinogen gene locus provides a unique system for the study of two-peaked day/night rhythms of gene expression in a synchronized form.

Animals↗
PubMed2003

Comparing gene expression profiles in human liver, gastric, and pancreatic tissues using full-length-enriched cDNA libraries.

In the post-genome-sequencing era, full-length cDNA-sequence resources are extremely useful for functional analyses of genes. In addition, comprehensive gene profiling of human tissues at the mRNA level is also useful in understanding the molecular mechanisms of tissue-specific functions and disease pathogenesis. In this study, to obtain a wide variety of full-length cDNA clones derived from digestive tissues, numerous expressed sequence tags were generated from libraries enriched with full-length cDNAs. In total, 13575 sequences were obtained from three cDNA libraries, which were constructed from tissues and cell lines of human liver, stomach, and pancreas. The integration of overlapping clones categorized the sequences into 5936 clusters (1666, 2746, and 2222 clusters in the liver, stomach, and pancreas, respectively). Of these, 1138 clones were scored as full-length cDNAs. Surprisingly, the redundant clones from all three tissues were assembled to show that only 101 genes (1.7% of the assembled 5936 genes) were shared. These results suggest that functional differences between tissues are probably related to their divergent gene expression profiles, and form a basis for understanding the molecular mechanisms underlying tissue-specific pathogenesis that are expressed in different organs. In addition, the full-length cDNAs obtained in this study should prove useful for future functional analyses of the genes expressed in digestive tissues.

Journal Article↗
PubMed2003

Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease.

To investigate the hepatitis B virus (HBV) genotype-related differences in the progression of liver disease, 585 patients with chronic HBV infection including 258 with histologically verified chronic liver disease (CLD) and 74 with hepatocellular carcinoma (HCC) were examined. The mean ages of both patients with advanced fibrosis (F3 or F4) and with HCC were significantly older in genotype B than in genotype C patients (P =.018, P =.024, respectively). Both the hepatitis B e antigen (HBeAg) negativity rate at biopsy and the cumulative HBe seroconversion rate in patients with CLD were significantly higher in genotype B patients than genotype C patients (P <.01, P =.022, respectively). Multivariate analysis revealed that genotype B, presence of precore mutation, high ALT levels, and severe histologic activity were independent factors for HBe seroconversion. Among all the biopsy-proven CLD patients, the ratio of patients with advanced fibrosis in genotype B was significantly lower than that in genotype C (4/30 vs. 74/224, respectively; P =.034). This difference was more remarkable in younger patients (< or =45 years; 1/25 vs. 47/180, respectively; P =.020), and there was no difference in older patients (>45 years). The distribution of each genotype between CLD and HCC was very similar (B and C: 11.2% and 87.0% vs. 10.8% and 89.2%, respectively). In conclusion, our results suggest that, although the patients with genotype B experience earlier HBe seroconversion, slower progression of liver fibrosis, and slower development of HCC, the life-long risk of progression to advanced fibrosis and development of HCC may not differ among genotypes B- and C-related chronic liver disease.

Adult↗
PubMed2003

In-house cDNA microarray analysis of gene expression profiles involved in SCC cell lines.

To analyze gene expression in oral cancer, we produced a specialized in-house cDNA microarray. The cDNA library was constructed from surgical specimens of oral squamous cell carcinoma (SCC) using an oligo-capping method. cDNA clones (n=4,800) were randomly selected and their 5'-end nucleotide sequences were determined. Overlapping clones were excluded, and 1,423 independent clones were selected and used for microarray production. Compared to the public nucleotide sequence database, 61% of our cDNA clones were full-length. By correlating expression patterns across SCC cell lines, we identified 53 genes (7 up-regulated and 46 down-regulated) that are differentially expressed in SCC cell lines compared to normal mucosa. Semi-quantitative RT-PCR analysis confirmed these findings and validity of our cDNA microarray. Using specimens from SCC patients, we investigated the expression status of the IL-1ra gene, which showed the down-regulated gene by microarray analysis. Gene expression clearly fell in the SCC specimens relative to their references, which indicated that our in-house cDNA microarray system rapidly identified and characterized candidate biomarkers for clinical use.

Carcinoma, Squamous Cell↗
PubMed2002

Identification of candidate genes for Sjögren's syndrome using MRL/lpr mouse model of Sjögren's syndrome and cDNA microarray analysis.

Sjögren's syndrome is a chronic autoimmune disease characterized by focal lymphocytic infiltration of lacrimal and salivary glands, but the precise mechanism of this syndrome is poorly understood. To clarify the mechanism of onset and progression of Sjögren's syndrome, it is necessary to identify Sjögren's syndrome-related genes. For this purpose, we used MLR/MpJ-lpr/lpr (MRL/lpr) mouse as a model of human secondary Sjögren's syndrome and analyzed specific mRNA expression pattern in MRL/lpr mouse salivary glands by in-house cDNA microarray. Among arrayed 2304 genes, 13 genes were isolated as highly expressed genes in MRL/lpr mouse salivary gland in comparison with MRL/MpJ-+/+ (MRL/+) mouse tissue. Subsequently, we performed RT-PCR analysis and confirmed the high expression level of nine genes; caspase3, Ly-6C.2, vimentin, Mel-14 antigen, cathepsin B, mpt1, Laptm5, Gnai2 and UCP2. Five of the nine genes have already been identified in patients with Sjögren's syndrome or mice models of the syndrome, but the remaining four genes; mpt1, Laptm5, Gnai2, and UCP2 have not been reported previously as Sjögren's syndrome-related genes. Although, further experiments are necessary to examine the relationship between these four genes and Sjögren's syndrome, our system of mouse model of Sjögren's syndrome combined with in-house cDNA microarray is suitable for the isolation of Sjögren's syndrome-related genes.

Animals↗
PubMed2002

Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor.

The low-density lipoprotein receptor-related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.

Animals↗
PubMed2002

Identification of the p33(ING1)-regulated genes that include cyclin B1 and proto-oncogene DEK by using cDNA microarray in a mouse mammary epithelial cell line NMuMG.

The candidate tumor suppressor p33(ING1) plays an important role in inducinggrowth arrest at G(0)-G(1) phase of the cell cycle and/or promoting apoptosis in cancerous cells. p33(ING1) is reported to act as a transcriptional cofactor by associating with tumor suppressor p53, HAT, or histone deacetyltransferase, suggesting that p33(ING1) is involved in chromatin-mediated transcriptional regulation. However, the molecular mechanism of p33(ING1)-mediated transcriptional regulation is poorly understood. Here we analyzed expression profiles in mouse mammary epithelial cells (NMuMG) by using a cDNA microarray consisting of 2304 mouse cDNAs after inducing transformation with antisense inhibitor of growth 1 (ING1) in retrovirus vector. The subsequent confirmation of the altered expression levels of the selected genes by semiquantitative reverse transcription-PCR demonstrated that overexpression of the antisense ING1 stimulated expression of 14 genes, which included cyclin B1, 12-O-tetradecanoylphorbol-13-acetate-inducible sequence 11, proto-oncogene DEK, and osteopontin, whereas we have detected transcriptional repression of 5 genes, including TPT1. In addition, adenovirus-mediated overexpression of ING1 in NMuMG cells resulted in down-regulation of cyclin B1, 12-O-tetradecanoylphorbol-13-acetate-inducible sequence 11, DEK, and osteopontin, whereas the levels of TPT1 expression were increased. The further analysis using p53(-/-) SAOS2 cells showed that the p33(ING1)-induced cyclin B1 down-regulation was p53 dependent. Thus, our cDNA microarray analysis suggested that p33(ING1) targets the multiple genes, including proto-oncogene DEK and cyclin B1, at least some of which are regulated in a p53-dependent manner, in the cells undergoing cell growth or apoptosis.

Animals↗
PubMed2002

Toxicogenomic effects of neonatal exposure to diethylstilbestrol on mouse testicular gene expression in the long term: a study using cDNA microarray analysis.

We examined the effect of neonatal exposure to diethylstilbestrol (DES) on mouse testicular gene expression, using in-house mouse fetus (day 14.5) cDNA microarrays. Newborn male ICR mice were exposed to DES (50 microg/mouse/day) from neonatal day 1 to 5. Differential expression was detected in 14 genes in 4-week-old (day 28) mouse testes by cDNA microarray analysis; 11 genes (AI035263, AU080565, AU080361, AU080678, AI131681, AU080631, AA986882, AI037066, AA986537, AI156816, and AI596237) were up-regulated and three genes (AI131656, AI118968, and AI117606) were down-regulated in DES-treated mouse testes. Higher expression levels of the former eight genes, out of the up-regulated genes picked-up by the microarray, were also confirmed by reverse transcription and real-time polymerase chain reaction (real-time RT-PCR) analysis. However, the differential expression of other genes could not be confirmed. Real-time RT-PCR analysis also revealed that expression levels of the eight genes were still higher in DES-treated testes at 8 and 12 weeks of age. Our results suggest that cDNA microarray analysis is a useful method by which a large number of gene expressions are simultaneously detected and changes in gene expression are screened. In addition, our results suggest that these genes, whose expressions are changed in the testes of adult mice by fetal or neonatal exposure to exogenous chemicals, might be candidates for predictive biological markers.

Animals↗
PubMed2002

Bisphenol a diglycidyl ether (BADGE) suppresses tumor necrosis factor-alpha production as a PPARgamma agonist in the murine macrophage-like cell line, RAW 264.7.

Bisphenol A diglycidyl ether (BADGE) is a newly described peroxisome proliferator-activated receptor gamma (PPARgamma) antagonist in adipogenic cells. In contrast, in the macrophage-like cell line RAW 264.7, BADGE, like the PPARgamma agonist pioglitazone hydrochloride, not only increased promoter activity of the PPARgamma-luciferase reporter gene, but also suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) production. These results suggest that BADGE is a PPARgamma agonist in RAW 264.7 cells. Furthermore, overexpression of the coactivator p300 restored BADGE- or pioglitazone hydrochloride-suppressed promoter activity of the nuclear factor-kappa B (NF-kappaB)-luciferase reporter gene, suggesting that PPARgamma may interfere with NF-kappaB transcriptional activity via coactivator competition.

Animals↗