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Objectives Epigenetic phenomena are changes in gene expression not involving the DNA sequence. DNA methylation is a major occurrence underlying epigenetic changes in human cells. Although aberrant DNA methylation is well documented in malignant lesions, limited information has been shown on the involvement of DNA methylation in oral lichen planus and oral lichenoid lesions (OLP). The present study aimed to investigate DNA methylation of E‐cadherin and p16 in OLP, and compare the findings with those in non‐inflamed gingiva (Non), radicular cyst (RC), and oral squamous cell carcinoma (SCC). Materials and methods Paraffin‐embedded surgical biopsy specimens were sliced, DNA was extracted, bisulfite treatment was applied, and methylation‐specific polymerase chain reaction was performed. Immunohistochemistry was performed to observe the relative expression patterns of these genes. Results E‐cadherin was hypermethylated in OLP (p

Bortezomib (Btz) is an active agent used to treat multiple myeloma (MM). Not all patients who receive Btz-containing therapy show a favorable response. Interaction of cellular adhesion molecules with MM and bone marrow stromal cells is crucial for the survival of MM cells. However, little is known about the role of these molecules in the sensitivity of MM to Btz-containing therapy. Thus, we evaluated the correlation between the level of cellular adhesion molecules in MM cells and the efficacy of Btz plus dexamethasone (Bd) therapy. The expression of the neural cell adhesion molecule gene (NCAM, also known as CD56), ITGA4, CXCR4, and other genes were analyzed in 74 samples of primary MM cells collected from patients before they received Bd therapy. Of the eight genes tested, expression of NCAM was lower among patients who responded poorly to Bd therapy. In vitro expression of NCAM induced by transfection of MM cells enhanced their sensitivity to Btz treatment by causing accumulation of polyubiquitinated proteins. Our results indicate that expression of NCAM is associated with better response to Btz treatment and is a promising candidate biomarker for predicting response to therapies involving Btz.

Hepatic stellate cells (HSCs) were reported to promote the progression of hepatocellular carcinoma (HCC), however its mechanism is uncertain. We previously reported that protein kinase R (PKR) in hepatocytes regulated HCC proliferation. In this study, we focused on the role of PKR in HSCs, and clarified the mechanism of its association with HCC progression. We confirmed the activation of PKR in a human HSC cell line (LX-2 cell). IL-1β is produced from HSCs stimulated by lipopolysaccharide (LPS) or palmitic acid which are likely activators of PKR in non-alcoholic steatohepatitis (NASH). Production was assessed by real-time PCR and ELISA. C16 and small interfering RNA (siRNA) were used to inhibit PKR in HSCs. The HCC cell line (HepG2 cell) was cultured with HSC conditioning medium to assess HCC progression, which was evaluated by proliferation and scratch assays. Expression of PKR was increased and activated in stimulated HSCs, and IL-1β production was also increased molecular. Key molecules of the mitogen-activated protein kinase pathway were also upregulated and activated by LPS. Otherwise, PKR inhibition by C16 and PKR siRNA decreased IL-1β production. HCC progression was promoted by HSC-stimulated conditioning medium although it was reduced by the conditioning medium from PKR-inhibited HSCs. Moreover, palmitic acid also upregulated IL-1β expression in HSCs, and conditioning medium from palmitic acid-stimulated HSCs promoted HCC proliferation. Stimulated HSCs by activators of PKR in NASH could play a role in promoting HCC progression through the production of IL-1β, via a mechanism that seems to be dependent on PKR activation.

Introduction Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies. Method and Results We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p

Advances in mouse neural circuit genetics, brain atlases, and behavioral assays provide a powerful system for modeling the genetic basis of cognition and psychiatric disease. However, a critical limitation of this approach is how to achieve concordance of mouse neurobiology with the ultimate goal of understanding the human brain. Previously, the common marmoset has shown promise as a genetic model system toward the linking of mouse and human studies. However, the advent of marmoset transgenic approaches will require an understanding of developmental principles in marmoset compared to mouse. In this study, we used gene expression analysis in marmoset brain to pose a series of fundamental questions on cortical development and evolution for direct comparison to existing mouse brain atlas expression data. Most genes showed reliable conservation of expression between marmoset and mouse. However, certain markers had strikingly divergent expression patterns. The lateral geniculate nucleus and pulvinar in the thalamus showed diversification of genetic organization between marmoset and mouse, suggesting they share some similarity. In contrast, gene expression patterns in early visual cortical areas showed marmoset-specific expression. In prefrontal cortex, some markers labeled architectonic areas and layers distinct between mouse and marmoset. Core hippocampus was conserved, while afferent areas showed divergence. Together, these results indicate that existing cortical areas are genetically conserved between marmoset and mouse, while differences in areal parcellation, afferent diversification, and layer complexity are associated with specific genes. Collectively, we propose that gene expression patterns in marmoset brain reveal important clues to the principles underlying the molecular evolution of cortical and cognitive expansion.

Purpose: We identified the endogenous retroviruses associated with TTYs (testis specific transcripts linked to the Y) in the AZFb region. We evaluated the relationship between endogenous retroviruses, and TTY expression patterns and function in spermatogenesis. Materials and Methods: We identified family members of TTYs in the AZFb region using computational screening. After investigating the relationship between the endogenous retrovirus genome and TTY expression patterns we screened genomic polymerase chain reaction products from TTY13 amplified from 790 Japanese men, including 275 with azoospermia, 285 with oligozoospermia and 230 who were fertile. Results: Computational screening revealed that 3 members of the TTY family, TTY9, 10 and 13, were regulated by endogenous retroviruses in the AZFb region. Homologous recombination between long terminal repeat of the TTY13 associated human endogenous retrovirus-K14C resulted in TTY13 deletion events. These deletions were more common in patients with azoospermia and oligozoospermia than in fertile males. Specifically 15.63% of the azoospermia group, 10.88% of the oligozoospermia group and 0% of fertile controls had only the deletion variant, indicating an association between the homologous recombination rate and the severity of spermatogenesis failure that was statistically significant (p

Summary Objective To investigate the mechanism of mechanical stress-induced expression and regulation of aggrecanases and examine the role of runt-related transcription factor 2 (RUNX-2) in chondrocyte-like cells. Methods SW1353 cells were seeded onto stretch chambers at a concentration of 5×10 4 cells/chamber, and a uni-axial cyclic tensile strain (CTS) (0.5Hz, 10% stretch) was applied for 30min. Total RNA was extracted, reverse transcribed, and analyzed by polymerase chain reaction (PCR) and real-time PCR. RUNX-2 overexpression and small interfering RNA (siRNA) targeting RUNX-2 were used to investigate the role of RUNX-2 in CTS-induced gene expression. The involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of RUNX-2, MMP-13 and ADAMTS-5 during CTS was examined by Western blotting. Results CTS induced expression of RUNX-2 , MMP-13 , ADAMTS-4 , -5 , and -9 . Overexpression of RUNX-2 up-regulated expression of MMP-13 and ADAMTS-5 , whereas RUNX-2 siRNA resulted in significant down-regulation of mechanically-induced MMP-13 and ADAMTS-5 expression. CTS induced activation of p38 MAPK, and CTS induction of RUNX-2 , MMP-13 and ADAMTS-5 mRNA was down-regulated by the selective p38 MAPK inhibitor SB203580 but not by the p44/42 MAPK inhibitor U0126, or the JNK MAPK inhibitor JNK inhibitor II. Conclusions RUNX-2 might have a role as a key downstream mediator of p38's ability to regulate mechanical stress-induced MMP-13 and ADAMTS-5 expression.

Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO₂ without any additional carbon or energy source. We performed a gene expression analysis of the oxidoreductases, which are involved in methanol metabolism, under various growth and induction conditions in N9T-4. A real-time PCR analysis revealed that the genes encoding NAD-dependent formaldehyde dehydrogenase (nFADH) and N,N'-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase (MDH) were strongly expressed under the oligotrophic conditions at levels of 20-100 fold those under heterotrophic conditions, in which n-tetradecane was used as the sole carbon source, while glucose did not affect the gene expression pattern in a minimum medium. The genes encoding mycothiol-dependent formaldehyde dehydrogenase (mFADH) and formate dehydrogenase were not induced under oligotrophic conditions, although mFADH expression was observed when formaldehyde was added to the induction medium. These results suggest that N9T-4 had three distinct formaldehyde oxidation systems, and that MDH and nFADH were the key enzymes in its oligotrophic growth.

Spermatogonial stem cells (SSCs) reside in undifferentiated type-A spermatogonia and contribute to continuous spermatogenesis by maintaining the balance between self-renewal and differentiation, thereby meeting the biological demand in the testis. Spermatogonia have to date been characterized principally through their morphology, but we herein report the detailed characterization of undifferentiated spermatogonia in mouse testes based on their gene expression profiles in combination with topological features. The detection of the germ cell-specific proteins Nanos2 and Nanos3 as markers of spermatogonia has enabled the clear dissection of complex populations of these cells as Nanos2 was recently shown to be involved in the maintenance of stem cells. Nanos2 is found to be almost exclusively expressed in A(s) to A(pr) cells, whereas Nanos3 is detectable in most undifferentiated spermatogonia (A(s) to A(al)) and differentiating A(1) spermatogonia. In our present study, we find that A(s) and A(pr) can be basically classified into three categories: (1) GFRalpha1(+)Nanos2(+)Nanos3(-)Ngn3(-), (2) GFRalpha1(+)Nanos2(+)Nanos3(+)Ngn3(-), and (3) GFRalpha1(-)Nanos2(+/-)Nanos3(+)Ngn3(+). We propose that the first of these groups is most likely to include the stem cell population and that Nanos3 may function in transit amplifying cells.

Purpose: It is important to identify the definitive molecular switches involved in the malignant transformation of premalignant tissues. Cellular senescence is a specific characteristic of precancerous tissues, but not of cancers, which might reflect tumorigenesis-protecting mechanisms in premalignant lesions. Polycomb protein Bmi1, which is a potent negative regulator of the p16INK4 gene, suppresses senescence in primary cells and is overexpressed in various cancers. We hypothesized that Bmi1 expression would also be dysregulated in precancerous lesions in human digestive precancerous tissues. Experimental Design: Bmi1 expression was investigated in cancerous and precancerous tissues of the digestive tract. The expression of p16, β-catenin, and Gli1 and the in vivo methylation status of the p16 gene were also analyzed in serial sections of colonic precancerous lesions. Results: Bmi1 was clearly overexpressed across a broad spectrum of gastrointestinal cancers, and the expression of Bmi1 increased in a manner that reflected the pathologic malignant features of precancerous colonic tissues (low-grade dysplasia, 12.9 ± 2.0%; high-grade dysplasia, 82.9 ± 1.6%; cancer, 87.5 ± 2.4%). p16 was also strongly expressed in high-grade dysplasia, but not in cancers. p16 promoter methylation was detected only in some Bmi1-positive neoplastic cells. Conclusions: Bmi1 overexpression was correlated with the malignant grades of human digestive precancerous tissues, which suggests that advanced Bmi1 dysregulation might predict malignant progression. The abnormal Bmi1 expression might link to malignant transformation via the disturbance of orderly histone modification.

Rebamipide, a gastromucosal protective drug, suppresses indomethacin-induced gastropathy in humans and rodents. Effects of rebamipide on gene expression in indomethacin-treated gastric mucosal cells (RGM1) were investigated using high-density oligonucleotide arrays. Indomethacin induced apoptosis in RGM1 cells in a dose-dependent manner. Rebamipide pretreatment significantly reduced indomethacin-induced apoptosis. We used gene expression profiling on high-density oligonucleotide probe arrays to characterize the transcriptional response of RGM1 cells to indomethacin treatment for 6 hr. Of the 8,799 probes examined, 717 (8.1%) were induced (400 probes) or repressed (317 probes) at least 1.5-fold. Among the 158 genes that were induced by indomethacin at least 2.0-fold, four genes that were down-regulated by rebamipide at least 2.0-fold are listed: growth arrest and DNA-damage-inducible 45 alpha (GADD 45 alpha), golgi SNAP receptor complex member 1, iodothyronine deiodinases, and transcription factor 8. Real time-PCR confirmed GADD 45 alpha expression and its inhibition by rebamipide. Inhibition of apoptosis-related genes is possibly important for the cytoprotective effect of rebamipide against indomethacin-induced gastric mucosal cell injury.

Interleukin (IL)-12 is a logical candidate for participation in the differentiation of T helper (Th) 1 cells. IL-12 is produced by macrophages and B cells and induces the production of interferon (IFN)-gamma from Th1 cells and NK cells. In this study, we show that the IFN-gamma itself is capable of inducing IL-12 mRNA expression in murine macrophage cell line J774. The mRNA was apparent by 12h after IFN-gamma treatment, although maximal induction required 36 to 48h. Furthermore, we investigated the signal transduction mechanism responsible for the mRNA expression. The IFN-gamma-inducible IL-12 mRNA was blocked by two protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, suggesting that PTK is involved in the IFN-gamma-inducible IL-12 mRNA expression.

Endotoxin-induced uveitis (EIU) is an animal model of acute ocular inflammation. Cytokines, chemokines, and nitric oxide (NO) have been reported to play important roles. We have determined whether heme oxygenase (HO)-1, a heat shock protein, can suppress EIU. EIU was induced by a footpad injection of lipopolysaccharide (LPS) in male Lewis rats. Hemin, an inducer of HO-1, was injected intraperitoneally 1 hr prior to the LPS injection. HO-1 and HO-2 expression in the iris-ciliary body (ICB) was studied by real time PCR and Western blot analysis. The number of infiltrating cells and the protein concentration in the aqueous humor (AqH) were evaluated by microscopy and by protein assay. The expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and IL-1beta mRNA was determined by real time PCR. The concentration of nitrate plus nitrite, and levels of IL-6 and TNF-alpha in the AqH were also evaluated by Griess reagents and by enzyme-linked immunosorbent assay, respectively. The expression of HO-1 mRNA and protein, induced by LPS, was enhanced significantly by pre-injection of hemin (P0.001). HO-2 was constitutively present in the ICB and was not up-regulated by LPS or by hemin. The number of infiltrating cells and the concentration of protein in the AqH was significantly elevated by LPS injection, and hemin significantly reduced the number of cells and the protein concentration (P0.0001). The expression of iNOS and IL-6 mRNA and protein were down-regulated by hemin (P0.001). Hemin is effective in inducing HO-1 and in reducing the ocular inflammation induced by LPS probably by down-regulating NO and pro-inflammatory cytokine expression.

We measured the relative expression levels of galectin-3 mRNA to beta-actin mRNA in normal thyroid tissues, thyroid tumor tissues and thyroid-derived fibroblasts. Galectin-3 mRNA was expressed ubiquitously in both benign and malignant thyroid tumors. Although a significant increase in its expression was observed in papillary carcinomas, no significant difference was observed between follicular carcinomas and adenomas. In contrast to the previous optimistic reports using immunohistochemical analysis of the galectin-3 protein expression, these results demonstrate that galectin-3 mRNA may not be a suitable target for molecular-based diagnosis of thyroid carcinomas.

In long-standing ulcerative colitis (UC), p53 mutations have been shown to occur by indirect detection methods such as PCR-SSCP. To clarify whether p53 gene mutations are early events in UC-associated neoplasia and to analyse clonality within dysplasia-associated lesions or masses (DALMs), the entire coding region of the p53 gene was analysed in DNA of microdissected single crypts by the polymerase chain reaction (PCR)-direct sequencing method. With a novel microdissection method using serial histological sections, the p53 gene (exons 2-11) was analysed in a total of 11 regenerative crypts and 76 single crypts within seven DALMs selected from three colectomy specimens of long-standing UC patients. Although p53 point mutations were found in at least one crypt in each DALM, heterogeneity in terms of the presence and the type of genetic change was marked, except in one carcinoma. As early events, p53 gene mutations were apparent even in some regenerative crypts (8/12 crypts). Some were of silent type. Altered p53 protein expression was confirmed in only 14/32 mutated crypts and was also evident in 24 other non-mutated examples by immunostaining of serial sections. Polyclonal p53 gene mutations were found in regenerative (REG) crypts and low-grade dysplasia (LGD), but monoclonal changes were noted in high-grade dysplasia (HGD) or carcinoma (Ca) in long-standing UC. At the single crypt level, however, p53 point mutations were not always linked to p53 overexpression, indicating a discrepancy between gene alteration and protein accumulation in LGD.

Two distinct human light subunits of the heteromeric amino acid transporter, y+LAT-1 coded by SLC7A7 and y+LAT-2 coded by SLC7A6, are both known to induce transport system y+L activity. SLC7A7 has already been identified as the gene responsible for lysinuric protein intolerance (LPI). We successfully identified five novel SLC7A7 variants (S238F, S489P, 1630delC, 1673delG, and IVS3-IVS5del9.7kb) in Japanese patients with LPI by PCR amplification and direct DNA sequencing. In addition, we performed a semi-quantitative expression analysis of SLC7A7 and SLC7A6 in human tissue. In normal tissue, the gene-expression ratio of SLC7A6 to SLC7A7 was high in the brain, muscle, and cultured skin fibroblasts; low in the kidneys and small intestine; and at an intermediate level in peripheral blood leukocytes, the lungs, and cultured lymphoblasts. The gene-expression ratio of SLC7A6 to SLC7A7 in cultured lymphoblasts was significantly different between normal subjects and LPI patients with R410X and/or S238F, where the relative amount of SLC7A7 mRNA was significantly lower and the relative amount of SLC7A6 mRNA was statistically higher in affected lymphoblasts than in normal cells. Expression of SLC7A7 and SLC7A6 may thus be interrelated in cultured lymphoblasts. Hum Mutat 20:375–381, 2002. © 2002 Wiley-Liss, Inc.

Triploid wheat hybrids between tetraploid wheat and Aegilops tauschii sometimes show abnormal growth phenotypes, and the growth abnormalities inhibit generation of wheat synthetic hexaploids. In type II necrosis, one of the growth abnormalities, necrotic cell death accompanied by marked growth repression occurs only under low temperature conditions. At normal temperature, the type II necrosis lines show grass-clump dwarfism with no necrotic symptoms, excess tillers, severe dwarfism and delayed flowering. Here, we report comparative expression analyses to elucidate the molecular mechanisms of the temperature-dependent phenotypic plasticity in the triploid wheat hybrids. We compared gene and small RNA expression profiles in crown tissues to characterize the temperature-dependent phenotypic plasticity. No up-regulation of defense-related genes was observed under the normal temperature, and down-regulation of wheat APETALA1-like MADS-box genes, considered to act as flowering promoters, was found in the grass-clump dwarf lines. Some microRNAs, including miR156, were up-regulated, whereas the levels of transcripts of the miR156 target genes SPLs, known to inhibit tiller and branch number, were reduced in crown tissues of the grass-clump dwarf lines at the normal temperature. Unusual expression of the miR156/SPLs module could explain the grass-clump dwarf phenotype. Dramatic alteration of gene expression profiles, including miRNA levels, in crown tissues is associated with the temperature-dependent phenotypic plasticity in type II necrosis/grass-clump dwarf wheat hybrids.

A series of nucleocapsid protein (NP)-deleted genes of the Onderstepoort strain was constructed in order to locate antigenic regions of the NP of canine distemper virus. The expression of proteins from 5'-deleted NP genes was examined in COS-7 cells by indirect immunofluorescence assay using three monoclonal antibodies (MAbs), c-5, f-5 and h-6, and a rabbit serum against NP. These MAbs reacted with two regions of NP. Amino acid residues from 1 to 80, and 337-358, were necessary and sufficient for formation of the epitopes identified by MAbs f-5 and h-6, and c-5, respectively. The proteins translated from intact or 3'-deleted genes were found to be localized in the nuclei of COS-7 cells, whereas the proteins from the 5'-deleted genes were mainly detected in the cytoplasm. These results suggested that 80 amino acid residues at the N-terminus are required for transportation of NP into the nucleus.

Using a polymerase chain reaction-mediated cloning procedure, we have identified a novel member, termed ANA (from Abundant in Neuroepithelium Area), of Tob/BTG1 family of antiproliferative genes. Molecular cloning and analysis of cDNAs revealed that the human and mouse ANA encoded a protein of 252 amino acids. The amino-terminal half of ANA was homologous to the previously characterized antiproliferative gene products, BTG1, PC3/TIS21/BTG2, and Tob. The human ANA gene was localized at chromosome 21q11.2-q21.1. ANA was expressed in a variety of tissues and cell lines, its expression being high in the ovary, testis, prostate, thymus, and lung. Further analysis revealed that ANA expression was high in the ventricular zone of the developing central nervous system. Finally, overexpression of ANA impaired serum-induced cell cycle progression from the G0/G1 to S phase. In conclusion, ANA is a fourth member of the Tob/BTG1 family that might play roles in neurogenesis in the central nervous system.

The physiological role of the prostaglandin E2 receptor EP4 subtype was investigated by generation of EP4-deficient-mice by gene targeting. Loss of the EP4 receptor was not lethal in utero, but most EP4 (-/-) neonates became pale and lethargic approximately 24 h after birth and died within 72 h. Less than 5% of the EP4 (-/-) mice survived and grew normally more than a year. Histological examination revealed that the ductus arteriosus in dead neonates remained open, while it was partially closed in the survivors. In situ hybridization study showed that EP4 mRNA was strongly expressed in the ductus. These results suggest that neonatal death is at least partly due to patent ductus arteriosus and that the EP4 receptor plays a role in regulation of the patency of this vessel. They also indicate that normal function of the EP4 receptor is essential in neonatal adaptation of the circulatory system.

A 2,048-bp nucleotide sequence containing a gene coding for an enzyme that degraded guar gum from Bacillus circulans K-1 was identified by polymerase chain reaction walking. This G-gene consisted of 1,551 nucleotides coding for a protein with Mr 55,242. The enzyme was overexpressed in Escherichia coli JM109 cells by the cloning the G-gene downstream of the lac Z promoter of pUC19. The molecular mass of recombinant G-enzyme estimated by SDS-PAGE was 62 KDa, close to that from strain K-1. Analysis of the recombinant enzyme showed GalNAc, Xyl, GlcNAc, Man, Glc, and Gal to account for 1.7%, 14.4%, 6.1%, 3.2%, 54.2%, and 10.4%, respectively, of the total monosaccharides. Polyacrylamide gel electrophoresis of this enzyme with staining gave a red band. The results suggested that the sugars accounted for the differences in the molecular masses. The recombinant enzyme had two kinds of N-terminal sequences, Thr-Met-Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile and Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile-Gly-Thr. Comparison of these sequences with the deduced N-terminal sequence coded for the G-gene showed that the amino acid, first Met, of the lac Z gene or the next residues Thr-Met in the recombinant enzyme were absent in the native enzyme. Methionines near and at the N-terminus of the mature protein probably were digested by methionine aminopeptidases of E. coli after translation. The properties of recombinant G-enzyme were similar to those of the enzyme from K-1 cells.

E1AF is a newly identified ets-oncogene family transcription factor. Previous reports have noted that E1AF can upregulate promoter activities of several matrix metalloproteinase (MMP) genes and showed that invasive potentials of oral squamous cell carcinoma-derived cell lines are correlated with expression of E1AF and MMPs. The invasive phenotype is restrained by transfection with an antisense E1AF expression vector. Thus, E1AF is thought to be highly correlated with malignant potentials of cancer cells. However, little is known about E1AF expression and cancer cell malignancies in in vivo tumours. In the present study, 27 oral squamous cell carcinoma (SCC) specimens were examined using RT-PCR, Southern blot hybridisation and in situ hybridisation (ISH) and compared to the clinicopathological parameters. Among the 27 patients, E1AF was detected in 15 cases. E1AF mRNA was detected in 13 of 17 invasive SCCs, whereas the majority of SCCs not expressing E1AF showed an expansive growth pattern. Increased prevalence of E1AF-positive oral SCC was observed in cases with nodal metastasis. These results indicate that E1AF may be involved in cancer cell malignancies through its ability to promote invasive potential.

We analyzed the genetic and epigenetic alterations of p16INK4A/CDKN2/MTS1 gene (MTS1 gene) in 38 primary urothelial cancers. Genetic alterations of the MTS1 gene consisted of one base substitution mutation in exon 2 (2.6%) and 6 homozygous deletions (16.2%). Hypermethylation of the 5' CpG island in exon 1 of the MTS1 gene was observed in 12 tumors (37.5%). Consequently, 19 of 38 tumors (50%) showed genetic alterations or epigenetic hypermethylation of the MTS1 gene. Retention of hypermethylated MTS1 gene(s) in 36% of the tumors showing loss of heterozygosity at the critical region indicates that the methylation modification could be an initial event followed by genomic rearrangements associated with total loss of MTS1 gene function. Immunohistochemical analysis of MTS1 expression revealed that all the tumors with genetic alterations of the MTS1 gene and 9 of 12 highly methylated tumors displayed an absence of MTS1 nuclear antigen. Genetic and epigenetic changes of the MTS1 gene were not correlated with the grade and stage of tumors, indicating that these alterations are early events in urothelial carcinogenesis, in which functional inactivation by hypermethylation is a predominant mechanism.

BACKGROUND Macrophage scavenger receptor (SR) is implicated in playing a key role in macrophage-derived foam cell formation by taking up a large amount of modified low-density lipoproteins (LDL). It has also been postulated that alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2MG/LRP) is involved in the development of foam cells by taking up apo E-enriched chylomicrons and VLDL remnants, and lipoprotein lipase-triglyceride-rich lipoprotein complexes. Accumulation of these lipid-loaded monocyte/ macrophages in the subendothelial space is considered to be an early event of atherogenesis. Since atherogenesis is considered to be accelerated in dialysis patients, we attempted to investigate whether gene expression of SR and alpha 2MG/LRP are altered in peripheral blood monocytes from patients on haemodialysis with a cuprophane (Cu) or polymethylmethacrylate (PMMA) membranes. METHODS Peripheral blood monocytes (PBM) were prepared from patients undergoing haemodialysis with a Cu membrane (n = 9), patients undergoing haemodialysis with a PMMA membrane (n = 9), and healthy controls (n = 7). In a separate experiment we examined SR gene expression in uraemic patients (n = 12) and healthy controls (n = 9). SR and alpha 2MG/LRP mRNA were semiquantitated using reverse-transcription polymerase chain reaction (RT-PCR) assay followed by Southern blotting. RESULTS SR mRNA expression in PBM from patients on chronic haemodialysis with a Cu membrane was about twofold higher than that in PBM from patients on chronic haemodialysis with a PMMA membrane or the controls (P < 0.05). alpha 2MG/LRP mRNA expression in PBM showed no difference among these, three groups. SR gene expression in monocytes from uraemic patients was not increased compared with that in the controls. CONCLUSION PBM from patients under Cu membrane dialysis showed higher gene expression of SR than patients under PMMA membrane dialysis, uraemic patients, or healthy controls. This increased gene expression of SR in monocytes may be associated with the pathogenesis of accelerated atherosclerosis in patients on dialysis with a Cu membrane.

Yoshida T, Hayashi M, Monkawa T, Saruta T. Regulation of obese mRNA expression by hormonal factors in primary cultures of rat adipocytes. Eur J Endocrinol 1996;135:619–25. ISSN 0804–4643 The obese (ob) gene has been cloned recently and its protein product is called "leptin". Leptin is an adipocyte-derived satiety factor that regulates body weight homeostasis. Several hormonal factors have been reported to regulate ob mRNA expression. To determine which factors are most important for regulation of ob mRNA expression, we examined the effects of insulin, dexamethasone, a β3-adrenergic agonist (CGP12177A), 8-bromo-cAMP, 8-bromo-cGMP and 1-methyl-3-isobutylxanthine (MIX) on primary cultured adipocytes. Rat adipocytes obtained from epididymal fat were cultured using the ceiling method. Total RNA was extracted and the expression of ob mRNA was measured by quantitative reverse transcription-polymerase chain reaction. After 24 h of incubation, 100 nmol/l insulin significantly increased the expression of ob mRNA (21.4-fold compared to control). Moreover, insulin increased ob mRNA expression in a dose-dependent manner over a range of 1–100 nmol/l. The effect of 100 nmol/l insulin was similar to that seen with 20% newborn calf serum. Dexamethasone (25–1000 nmol/l) also increased ob mRNA expression (2.5–2.9-fold). The effect of dexamethasone occurred more rapidly than insulin. CGP12177A (1–10 μmol/l) and 0.5 mmol/l 8-bromo-cAMP had no effects, whereas 0.5 mmol/l 8-bromo-cGMP and 0.5 mmol/l MIX had stimulatory effects (2.8- and 2.4-fold increase in ob mRNA, respectively). The combination of 250 nmol/l dexamethasone and 0.5 mmol/l MIX did not have an additive effect on ob mRNA levels. Our present data suggest that, of these agents, insulin is the most important factor regulating ob mRNA expression. Matsuhiko Hayashi, Department of Internal Medicine, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan

Cyclic ADP-ribose (cADPR), a well-known stimulator of ca(2+) release from the intracellular Ca(2+) pool, has recently emerged as a potential regulator of insulin secretion in pancreatic beta cells. As recently described, BST-1 is a glycosyl-phosphatidylinositol (GPI)-anchored surface molecule that exhibits homology with CD38 and Aplysia ADP-ribosyl cyclase. Like CD38, BST-1 has both ADP-ribosyl cyclase and cADPR hydrolase activities. As a step toward elucidating the physiological role of cADPR in insulin secretion we examined whether BST-1 is expressed in pancreatic islet cells. Sensitive reverse transcription-polymerase chain reaction detected almost as abundant expression of BST-1 mRNA in pancreatic islets as CD38 mRNA. Immunohistochemical analyses utilizing mirror image sections revealed that BST-1 protein is expressed in a majority of the cells in pancreatic islets and that at least beta cells and, to an even greater extent, alpha cells express BST-1. These observations suggest the involvement of multiple enzymes in the regulation of cADPR concentrations in pancreatic islet cells.

Epigenetic alterations such as genomic imprinting might play an important role in human tumorigenesis, in addition to specific genetic alterations. To clarify the role of genetic and/or epigenetic alterations in the tumorigenesis of testicular germ cell tumors (GCTs), we analyzed 40 primary and 3 metastatic testicular GCTs with regard to specific chromosomal losses and their parental origin. A high incidence of loss of heterozygosity (LOH) was demonstrated on chromosomes 1p, 3p, 11p, and 17p: 9/19 (47%), 18/39 (46%), 13/40 (33%) and 20/36 (56%), respectively. However, there was no correlation between the frequency of LOH on any chromosome and clinicopathological features. Regarding the parental origin of the lost allele at these chromosomes, preferential loss was not demonstrated in this study. To clarify the imprinting status in GCTs, we analyzed the allele-specific expression of the H19 gene, which is paternally imprinted on chromosome 11p. All of 11 tumors without LOH at this locus showed biallelic expression of H19. Based on previous work demonstrating the biallelic expression of H19 in primordial germ cells and spermatogonia in the mouse germ line, these results suggest that the biallelic expression of H19 in testicular GCTs reflects the characteristics of the original germ cells in which the imprinting marking has been erased and not established, rather than loss of imprinting during tumorigenesis. It is also possible that a failure to re-establish the imprinting might be an initial event which leads to testicular GCTs.

This brief overview indicates that a large amount of intriguing information has emerged in the last few years about the unusual activity of the CD44 gene in neoplasia. It is a large gene and potentially could produce the most alternative splicing combinations yet described for any locus [1]. Although it is not yet known whether some of these are biologically inadmissable, several different isoforms containing various exon combinations have so far been detected. Therefore, it is likely that unravelling whether the observed disturbances in its regulation are causally involved in tumour induction and/or progression, or are merely consequences of other events, will take much time and effort.

"Gene trapping" in embryonic stem (ES) cells is a novel approach to identify a series of genes in mammals concomitant with the production of the corresponding mutant mice. However, this approach is currently unable to identify genes that are not expressed in ES cells. Here we describe a strategy to identify gene trapping clones which is not based on expression of a reporter gene. It uses the neor gene which lacks a polyadenylation signal and has a splice donor signal. Expression of the neor gene as fusion transcripts with the 3' end containing the polyadenylation signal of tagged genes allows the identification of these clones by 3' rapid amplification of the cDNA end in undifferentiated ES cells, even if the genes are not expressed in ES cells. Amplification was observed in about 25% of G418-resistant clones. Sequence analyses suggested the amplifications represent gene trapping events. The feasibility of this approach was further assessed by analysing one clone, PAT-12, in detail.

Mutations in the dystrophin gene cause the X chromosome-linked, recessive Duchenne and Becker muscular dystrophies. Dystrophin, a large cytoskeletal protein, copurifies with a complex of dystrophin-associated proteins which serve to anchor dystrophin to the sarcolemma. One of these associated proteins, adhalin, has been implicated as a candidate for severe childhood autosomal recessive muscular dystrophy (SCARMD) due to absence of anti-adhalin staining in muscle biopsy samples taken from SCARMD patients. Furthermore, the Duchenne-like dystrophic phenotype seen in the SCARMD families was shown to be tightly linked to chromosome 13 markers. To determine the genetic mutation responsible for autosomal dystrophy, we characterized the human adhalin gene. Contrary to our expectation, human adhalin was mapped to chromosome 17q21, excluding adhalin as the gene causing chromosome 13-associated SCARMD. Additionally, a splice form of adhalin message was found that predicts a 35-kDa nontransmembrane adhalin. The expression of both adhalin splice forms is exclusively restricted to striated muscle, unlike other components of the dystrophin-glycoprotein complex.

Taking advantage of the reverse transcriptase-polymerase chain reaction (RT-PCR), we have analyzed T cell receptor gamma-chain mRNA of synovial fluid gamma/delta T cells from patients with rheumatoid arthritis (RA) in comparison with those of peripheral blood mononuclear cells (PBMC) from RA patients and healthy individuals. The quantitative RT-PCR method in conjunction with nucleotide sequencing revealed the frequent usage of the V gamma 3 gene segment in RA synovial fluid mononuclear cells (SFMC) (p < 0.01) which in PBMC of healthy individuals occurred rarely. PBMC of most healthy individuals expressed the V gamma 9 gene predominantly (p < 0.01) as expected. However, only half of RA patients showed elevated levels of the V gamma 9 gene expression in their PBMC. The gamma-chain mRNA containing the V gamma 3 gene in RA SFMC showed no conserved junctional sequence (complementarity-determining region 3). To investigate the nature of ligands recognized by the V gamma 3-bearing T cells, we analyzed V gamma gene usage of RA SFMC, RA PBMC, and normal PBMC stimulated with Mycobacterium tuberculosis (MT) or MT plus interleukin-2 since there is mounting evidence of high reactivity of RA SFMC to MT and mycobacterial heat-shock protein 65. However, the V gamma usage appeared to be mostly V gamma 9 in RA SFMC, RA PBMC and normal PBMC. Taken together these results suggest that an as yet unknown antigen(s) (other than MT) might select gamma/delta T cells expressing the V gamma 3 gene in RA SFMC.

In the developing mammalian central nervous system, neural precursor cells show a tightly regulated inverse relationship between cell proliferation and differentiation. The molecular mechanisms which control the inter-relationship between these two events are poorly understood. To approach this problem, we previously identified several novel genes which are most prominently expressed in the early embryonic brain. Further cloning and sequencing of one such gene, Nedd1, revealed that it can encode a protein with a M(r) of 71,000, the amino-terminal half of which shares significant structural similarity with the beta-subunit of heterotrimeric GTP-binding proteins. Nedd1 mRNA is strongly expressed in early embryonic brain, but it can be detected at low levels in a number of adult tissues as well as cell lines and is up-regulated in an embryonal carcinoma cell line upon retinoic acid-induced differentiation. Ectopic expression of Nedd1 gene by means of eukaryotic vectors in various cell lines resulted in varying degrees of growth suppression. The strongest effects were evident in differentiation-competent neuroblastoma-derived cell lines. Our results suggest that the Nedd1 gene may play a role in the differentiation-coupled growth arrest in neuronal cells.

Using subcutaneous solid tumors produced by U251-MG human glioma cells, we studied the in vivo transfection of the cells with the tumor necrosis factor-alpha (TNF-alpha) gene delivered by means of liposomes. When the tumor had become 7 mm in diameter, liposomes with entrapped TNF-alpha gene were injected into the center of the subcutaneous tumor. We found that mRNA of transfection-induced TNF-alpha, which was expressed in the tumor tissue, was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) method and its protein was demonstrated by enzyme-linked immunoassay. Growth of the tumor was inhibited when the injection was carried out five times at every other day. The growth-inhibitory effect by transfection-induced TNF-alpha was much remarkable as compared with exogenous TNF-alpha and the effect was enhanced by the intraperitoneal injection of gamma-interferon (IFN-gamma) 12 h prior to intratumoral injection of the liposomes.

Background Scavenger receptors are generally expressed in macrophages and vascular endothelial cells and some scavenger receptors are thought to contribute to the development of atherosclerosis. Methods We cloned the cDNA of a zebrafish CL-P1 (collectin placenta 1) and performed a knockdown study using its antisense morpholino oligonucleotides (MO). Results Zebrafish CL-P1 (zCL-P1) is 51% identical to human CL-P1 in its amino acid sequence. Microbes and OxLDL bound to zCL-P1 cDNA transfected cells. zCL-P1 mRNA expression gradually increased after 6 hours post-fertilization (hpf), reached its highest level at 24 hpf, and then decreased, which is similar to the gene expression pattern of Tie-2. The knockdown of zCL-P1 led to an increase in the number of zebrafish embryos with severe morphological abnormalities such as short body lengths and defects in the dorsal aorta at 48 hpf. Simultaneous injection of both MO and synthetic zCL-P1 or zVEGF mRNA rescued the abnormal phenotype. Conclusions In vivo knockdown study shows that zCL-P1 is implicated in vasculogenesis and those of our in vitro study support its role as a scavenger receptor. General significance These results suggest that zCL-P1 might be essential for vasculogenesis during the early embryonic phase in bone fish.

We have detected a potent gelatinolytic activity in the culture supernatant of a metastatic tumor line, SN-H, derived from a murine squamous cell carcinoma. The relative molecular weight of the gelatinase was estimated as 105-kDa by gelatin zymography. We have cloned the cDNA of this gelatinase and the 3160-bp sequence has been determined. From the translated amino acid sequence, the positions of the cysteine residues and the functional domain structure are highly homologous to the human 92-kDa gelatinase. The nucleotide and amino acid sequence homology between these two cDNAs are 75% and 72%, respectively. Transfection of the cDNA in an expression vector resulted in production of the 105-kDa gelatinase, thus confirming that this cDNA is functional.

To identify molecular therapeutic targets for glioma, we performed gene expression profiling by using a complementary DNA (cDNA) microarray method and identified the urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180) as a gene expressed highly in glioma tissue compared with the normal brain tissue. The uPARAP is an endocytic receptor for collagen. In certain cell types, uPARAP occurs in a complex with the urokinase plasminogen activator receptor (uPAR) where it fulfills other functions in addition to collagenolysis. Quantitative PCR analysis using a cDNA panel revealed higher expression levels of uPARAP in glioma tissue compared with normal brain tissue. Western blot analysis revealed that the uPARAP protein was expressed in glioma samples and two glioma cell lines, KNS42 and KNS81, but not expressed in control tissue from the normal brain. Introduction of small interfering RNA-targeted uPARAP into the two different glioma cell lines, KNS42 and KNS81, resulted in downregulation of uPARAP expression, and it significantly suppressed glioma cell migration and invasion in vitro. Control glioma cells showed small cell bodies, whereas uPARAP siRNA-treated glioma cells exhibited large and flat morphology. Most of the polymeric actin in the control glioma cells was concentrated in the lamellipodia that are observed in mobile cells. In contrast, in the uPARAP siRNA-treated glioma cells, polymeric actin became organized in stress fibers and the lamellipodia disappeared, characteristic of immobile cells. Our present study suggests that uPARAP may be involved in glioma cell invasiveness through actin cytoskeletal rearrangement. downregulation of uPARAP may be a novel anti-invasion therapeutic strategy for malignant gliomas.

Gene expression of platelet-derived growth factor (PDGF) and its receptors in cultured human retinal pigment epithelial (RPE) cells was studied by using semiquantitative polymerase chain reaction. The RPE cells were found to express PDGF A- and B-chain genes as well as alpha- and beta-receptor genes with dominant expression of B-chain and beta-receptor isoforms. Phorbol myristate acetate (PMA) and thrombin increased the expression of PDGF B-chain gene to 19.8 +/- 1.75 and 15.9 +/- 1.84 fold (n = 3) of the control without affecting beta-receptor gene expression. PDGF produced by the RPE cells may play an important role in the pathogenesis of some ocular proliferative diseases.

Infiltrating T cells around salivary glands in the lips of Sjogren's syndrome (SjS) patients are crucial in the pathogenesis of this disease. To analyze the nature of infiltrating T cells, their T cell receptor repertoire was examined with quantitative polymerase chain reaction. The repertoire of V beta transcripts in lips of SjS was not restricted; however, the V beta 2 and V beta 13 genes were predominantly expressed on the T cells of lip specimens in six and four of seven lips, respectively. Predominance of these genes was specific in lips because no predominant V beta transcripts were found in lips from healthy subjects and PBLs from SjS patients. These results indicated that the V beta 2- and V beta 13-positive T cells expanded specifically and preferentially in SjS lips, thereby suggesting the possible role in triggering the autoimmunity of this disease.

We isolated two rice endo-(1,3;1,4)-beta-glucanase genes, denoted OsEGL1 and OsEGL2, which encoded proteins that shared 64% amino acid sequence identity. Both the OsEGL1 and OsEGL2 genes were successfully expressed in Escherichia coli to produce functional proteins. Purified OsEGL1 and OsEGL2 proteins hydrolyzed (1,3;1,4)-beta-glucans, but not (1,3;1,6)-beta-linked or (1,3)-beta-linked glucopolysaccharides nor carboxymethyl cellulose, similar to previously characterized grass endo-(1,3;1,4)-beta-glucanases. RNA blot analysis revealed that the OsEGL1 gene is expressed constitutively not only in young roots of rice seedlings, but also in mature roots of adult rice plants. Little or no expression of the OsEGL2 gene was observed in all tissues or treatments tested, but database and RT-PCR analysis indicated it is expressed in ripening panicle. In rice seedling leaves, OsEGL1 gene expression significantly increased in response to methyl jasmonate, abscisic acid, ethephon and mechanical wounding. Mechanical wounding also increased the leaf elongation rate in rice seedlings by 16% relative to that of control seedlings at day 4 after treatment. The increase in the leaf elongation rate of rice seedlings treated under mechanical wounding was concomitant with an increase in OsEGL1 expression levels in seedling leaves.

This study analyzed T-lymphocyte-receptor V beta genes of infiltrating lymphocytes into the pancreases of 12- to 16-wk-old NOD mice with severe insulitis and 5-wk-old mice with mild insulitis by the quantitative polymerase chain reaction method. The V beta transcripts on infiltrating T lymphocytes into pancreases with severe insulitis in older NOD mice were diverse. In contrast, the V beta 11 gene transcript was predominantly expressed on T lymphocytes in the pancreas in younger NOD mice with mild insulitis, suggesting the possible role of V beta 11+ T lymphocytes in triggering insulitis in this species.

Monocyte chemoattractant protein-1 (MCP-1) is a 76-amino-acid chemokine that is considered to be an important chemotactic factor for monocytes. MCP-1 is expressed in the macrophage-rich areas of atherosclerotic lesions. A recent report indicated that MCP-1 expression in human umbilical vein endothelial cells (HUVECs) is induced by the stimulation of tumor necrosis factor (TNF)-alpha via the c-Jun N-terminal kinases (JNK) pathway. In this study, we examined the effects of JNK inhibitor (JNKI-1), on MCP-1 expression. The results of this study indicated that the expression of MCP-1 mRNA and protein were stimulated in the presence of TNF-alpha. TNF-alpha stimulated the phosphrylation of JNK, however, JNKI-1 inhibited the TNF-alpha stimulated MCP-1 secretion and gene expression. As expected, JNKI-1 blocked the stimulatory effect of TNF-alpha on the MCP-1 promoter activity. In conclusion, JNKI-1 partially inhibits the TNF-alpha-induced MCP-1 expression in HUVECs, and therefore JNKI-1 may be of therapeutic value in the treatment of diseases such as atherosclerosis.