Hisao Ueyama

Anti-tumor-promoting activity of derivatives of abieslactone, a natural triterpenoid isolated from several Abies genus.

Abiesenonic acid methyl ester (AVB-I acid methyl ester), a triterpenoid compound prepared from abieslactone, suppressed tumor promoter-induced phenomena in vitro and in vivo; i.e., AVB-I acid methyl ester inhibited 12-o-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32Pi-incorporation into phospholipids of cultured cells and the promoting action of TPA on skin tumor formation in mice initiated with 7,12-dimethylbenz[a]anthracene.

Cloning and nucleotide sequence of a human Zn-α2-glycoprotein cDNA and chromosomal assignment of its gene

A cDNA clone of Zn-alpha 2-glycoprotein (Zn alpha 2gp) was isolated from a human prostate library. The amino acid sequence of prostate Zn alpha 2gp deduced from the nucleotide sequence was identical to the one previously reported on the Zn alpha 2gp protein purified from human blood plasma, except at three positions: the 65th and 222nd amino acid residues were Gln (----Glu) and Glu (----Gln), and there was a two amino acid insertion (Ile-Phe) between the 75th (Glu) and 76th (Met) amino acids. Southern blot analysis of human genomic DNA, however, suggested a single gene encoding Zn alpha 2gp. Using a panel of rodent-human somatic cell hybrids, the Zn alpha 2 gp gene was assigned to human chromosome 7.

Polymorphic CAG repeats of the androgen receptor gene and rheumatoid arthritis

OBJECTIVE In view of the possible role of androgens in the pathogenesis of rheumatoid arthritis (RA), this study investigated the association between repeat lengths of CAG microsatellites of the androgen receptor (AR) gene and RA. METHODS The number of CAG repeats in exon 1 of the AR gene was determined in 90 men and 276 women with RA, as well as in 305 male and 332 female controls. RESULTS The male RA patients tended to have shorter repeats than the male controls (22.5 versus 23.1, p=0.07), whereas the female RA patients had similar repeats to the female controls (22.7 versus 22.9, p=0.17). Patients of both sexes were divided into younger and older age at onset groups, and compared with younger and older controls. Younger onset male RA patients had significantly shorter CAG repeat lengths than the younger male controls (21.8 versus 23.2, p=0.007) or the older onset male RA patients (21.8 versus 23.2, p=0.04). Older onset male RA and both younger and older onset female RA patients had similar CAG repeat lengths when compared with their controls. Neither seropositivity nor rheumatoid nodule positivity had a significant relation with CAG repeat lengths. CONCLUSION Shorter CAG repeats of the AR gene, presenting high levels of transactivation activity, are related to younger age onset male RA, suggesting the possible role of androgens as a modulating factor.

Novel KCNE3 mutation reduces repolarizing potassium current and associated with long QT syndrome.

Long QT syndrome (LQTS) is an inherited disease involving mutations in the genes encoding a number of cardiac ion channels and a membrane adaptor protein. Among the genes that are responsible for LQTS, KCNE1 and KCNE2 are members of the KCNE family of genes, and function as ancillary subunits of Kv channels. The third KCNE gene, KCNE3, is expressed in cardiac myocytes and interacts with KCNQ1 to change the channel properties. However, KCNE3 has never been linked to LQTS. To investigate the association between KCNE3 and LQTS, we conducted a genetic screening of KCNE3 mutations and single nucleotide polymorphisms (SNPs) in 485 Japanese LQTS probands using DHPLC‐WAVE system and direct sequencing. Consequently, we identified two KCNE3 missense mutations, located in the N‐ and C‐terminal domains. The functional effects of these mutations were examined by heterologous expression systems using CHO cells stably expressing KCNQ1. One mutation, p.R99λH was identified in a 76‐year‐old woman who suffered torsades de pointes (TdP) after administration of disopyramide. Another mutation, p.T4A was identified in a 16‐year‐old boy and 67‐year‐old woman. Although the boy carried another KCNH2 mutation, he was asymptomatic. On the other hand, the woman suffered from hypokalemia‐induced TdP. In a series of electrophysiological analyses, the KCNQ1(Q1)+KCNE3(E3)‐R99λH channel significantly reduced outward current compared to Q1+E3‐WT, though the current density of the Q1+E3‐T4A channel displayed no statistical significance. This is the first report of KCNE3 mutations associated with LQTS. Screening for variants in the KCNE3 gene is of clinical importance for LQTS patients. Hum Mutat 30, 557–563, 2009.

Unique haplotype in exon 3 of cone opsin mRNA affects splicing of its precursor, leading to congenital color vision defect.

We have analyzed L/M visual pigment gene arrays in 119 Japanese men with protanopia color vision defect and found that five had a normal gene order of L-M. Among the five men, two (identified as A376 and A642) had apparently normal L genes. To clarify their L gene defect, the whole L or M gene from A376 and control subjects was cloned in an expression vector. Total RNA extracted from the transfected HEK293 cells was analyzed by Northern blot and reverse transcription-polymerase chain reaction. The product from the cloned L gene of A376 was smaller than the normal control due to the absence of exon 3. To investigate such exon-skipping at splicing, minigenes of exon 3 accompanying introns 2 and 3 were prepared from A376, A642, and control subjects. The minigenes of A376 (L) and A642 (L) showed the product lacking exon 3 only, while the minigene of normal control N44 (L) showed the product retaining exon 3 only. Exchanging of introns 2 and 3 between the A376 (L) and N44 (L) minigenes showed that the skipping of exon 3 was caused by the exon itself. Seven differences in exon 3 between A376 (L) and N44 (L) were all within already-known polymorphisms as follows: G(151-3), C(153-1), G(155-3), A(171-1), T(171-3), G(178-1) and G(180-1) in A376 (L) and A642 (L), and A(151-3), A(153-1), C(155-3), G(171-1), G(171-3), A(178-1) and T(180-1) in N44 (L). An in vitro mutagenesis experiment with these nucleotides in the minigenes showed that exon 3 was completely skipped at splicing only in the haplotype observed in A376 (L) and A642 (L). These results suggest that complete skipping of exon 3 at splicing, due to the unique haplotype of the exon, causes loss of expression of L-opsin in these men.

Localization of Urokinase‐type Plasminogen Activator, Plasminogen Activator Inhibitor‐1, 2 and Plasminogen in Colon Cancer

We examined the localization of urokinase‐type plasminogen activator (u‐PA), plasminogen activator inhibitors (PAI‐1 and PAI‐2) and plasminogen (plg) in 26 cases of colon cancer by immunohistochemical staining. The u‐PA antigen was detected in the cytoplasm of cancer cells (18/26) and stromal cells adjacent to cancer tissues (9/26). The localization of u‐PA mRNA examined by in situ hybridization was consistent with that of u‐PA antigen. The PAI‐1 antigen was detected in fibroblasts and endothelial cells (22/26), while PAI‐2 antigen was found in cancer cells (20/26). The plg antigen was seen in the extracellular matrix of the cancer stroma. The u‐PA expression in cancer cells was significantly more frequently detected in cases with lymph node metastasis than in cases without metastasis. In either PAI‐1‐ or PAI‐2‐expressing cases, lymph node metastasis seemed to be restrained. These findings indicate that cancer cells themselves produce u‐PA, and suggest that u‐PA converts plg into plasmin, which dissolves the extracellular matrix surrounding cancer cells, resulting in cancer invasion and metastasis. PAI‐1 and PAI‐2 may have inhibitory actions on cancer invasion and metastasis mediated by u‐PA.

Number and variations of the red and green visual pigment genes in Japanese men with normal color vision

PURPOSE We analyzed the red/green visual pigment genes in color-normal Japanese men to understand the relationship between color anomalies and genetic defects. METHODS DNA from 120 color-normal Japanese men was subjected to polymerase chain reaction (PCR)-amplification for exons 2-5 of the red/green visual pigment genes and the PCR products were sequenced. The red:green gene ratios were estimated from the sequencing electropherograms of exon 5 and also from MvaI-restriction fragment analysis of the same exon. The first gene and the downstream genes in the pigment gene array were separately analyzed by PCR, direct sequencing, and/or single-strand conformation polymorphisms. RESULTS The red:green gene ratios estimated from the ratios of peak heights of nucleotides on the sequencing electropherograms coincided with those estimated from the MvaI-restriction fragment analysis. Among the subjects analyzed, they were 1:1 in 43% (n = 52), 1:2 in 41% (n = 49), 1:3 in 6% (n = 7), and 1:>3 in 9% (n = 11). The first gene in the pigment gene arrays was red in all subjects. Only 1 subject (N22) had a green-red hybrid gene. Exons 2 and 4 had 2 haplotypes each, but exon 3 was highly polymorphic. Exon 5 of the green genes had one polymorphism at codon 283 with a frequency of 32%. CONCLUSIONS The features of visual pigment genes in color-normal Japanese men were revealed. The data and establishing techniques may be useful for analyzing these genes in color-deficient subjects in the Japanese population.

Three Different Cone Opsin Gene Array Mutational Mechanisms with Genotype–Phenotype Correlation and Functional Investigation of Cone Opsin Variants

Mutations in the OPN1LW (L‐) and OPN1MW (M‐)cone opsin genes underlie a spectrum of cone photoreceptor defects from stationary loss of color vision to progressive retinal degeneration. Genotypes of 22 families with a range of cone disorders were grouped into three classes: deletions of the locus control region (LCR); missense mutation (p.Cys203Arg) in an L‐/M‐hybrid gene; and exon 3 single‐nucleotide polymorphism (SNP) interchange haplotypes in an otherwise normal gene array. Moderate‐to‐high myopia was observed in all mutation categories. Individuals with LCR deletions or p.Cys203Arg mutations were more likely to have nystagmus and poor vision, with disease progression in some p.Cys203Arg patients. Three disease‐associated exon 3 SNP haplotypes encoding LIAVA, LVAVA, or MIAVA were identified in our cohort. These patients were less likely to have nystagmus but more likely to show progression, with all patients over the age of 40 years having marked macular abnormalities. Previously, the haplotype LIAVA has been shown to result in exon 3 skipping. Here, we show that haplotypes LVAVA and MIAVA also result in aberrant splicing, with a residual low level of correctly spliced cone opsin. The OPN1LW/OPN1MW:c.532A>G SNP, common to all three disease‐associated haplotypes, appears to be principally responsible for this mutational mechanism.

Effect of dynamic compressive loading and its combination with a growth factor on the chondrocytic phenotype of 3-dimensional scaffold-embedded chondrocytes

Background and purpose Three-dimensionally (3D-) embedded chondrocytes have been suggested to maintain the chondrocytic phenotype. Furthermore, mechanical stress and growth factors have been found to be capable of enhancing cell proliferation and ECM synthesis. We investigated the effect of mechanical loading and growth factors on reactivation of the 3D-embedded chondrocytes. Methods Freshly isolated chondrocytes from rat articular cartilage were grown in monolayer cultures and then in collagen gel. Real-time RT-PCR and histological analysis for aggrecan and type II and type I collagen was performed to evaluate their chondrocytic activity. Then, the 3D-embedded chondrocytes were cultured under either mechanical loading alone or in combination with growth factor. The dynamic compression (5% compression, 0.33 Hz) was loaded for 4 durations: 0, 10, 60, and 120 min/day. The growth factor administered was either basic fibroblast growth factor (bFGF) or bone morphogenetic protein-2 (BMP-2). Results Mechanical loading statistically significantly reactivated the aggrecan and type II collagen expression with loading of 60 min/day as compared to the other durations. The presence of BMP-2 and bFGF clearly enhanced the aggrecan and type II collagen expression of 3D-embedded chondrocytes. Unlike previous reports using monolayer chondrocytes, however, BMP-2 or bFGF did not augment the chondrocytic phenotype when applied together with mechanical loading. Interpretation Dynamic compression effectively reactivated the dedifferentiated chondrocytes in 3D culture. However, the growth factors did not play any synergistic role when applied with dynamic compressive loading, suggesting that growth factors should be administered at different time points during regeneration of the transplantation-ready cartilage.

Assignment of the vascular smooth muscle actin gene ACTSA to human chromosome 10

SummaryHuman vascular smooth muscle actin gene (ACTSA) was cloned and its unique sequence was used as the hybridization probe for Southern blot analysis of DNAs from 18 rodent-human somatic cell hybrids; the gene was assigned to human chromosome 10. Regional mapping by in situ hybridization showed that the gene is located on the long arm (q22–q24) of the chromosome. Thus, the gene is on a different chromosome from the other four actin genes so far examined.