Search

Blue cone monochromacy (BCM) is characterized by loss of function of both OPN1LW (the first) and OPN1MW (the downstream) genes on the X chromosome. The purpose of this study was to investigate the first and downstream genes in the OPN1LW/OPN1MW array in four unrelated Japanese males with BCM. In Case 1, only one gene was present. Abnormalities were found in the promoter, which had a mixed unique profile of first and downstream gene promoters and a −71A > C substitution. As the promoter was active in the reporter assay, the cause of BCM remains unclear. In Case 2, the same novel mutation, M273K, was present in exon 5 of both genes in a two-gene array. The mutant pigments showed no absorbance at any of the wavelengths tested, suggesting that the mutation causes pigment dysfunction. Case 3 had a large deletion including the locus control region and entire first gene. Case 4 also had a large deletion involving exons 2–6 of the first gene. As an intact LCR was present upstream and one apparently normal downstream gene was present, BCM in Case 4 was not ascribed solely to the deletion. The deletions in Cases 3 and 4 were considered to have been caused by non-homologous recombination.

To present the characteristics and pathology of a patient with congenital achromatopsia. The patient was a 22-year-old Japanese woman who was 8 years old when she first visited our clinic. Comprehensive ophthalmic examinations including visual acuity measurements, perimetry, optical coherence tomography (OCT), fundus autofluorescence (FAF) imaging, electroretinography (ERG), and color vision tests were performed. Her genomic DNA was used as the template for the amplification of exons of five candidate genes for achromatopsia; CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H, and the amplified products were sequenced. A missense mutation, found in the CNGA3, was studied both electrophysiologically and biochemically. Her phenotype was typical of congenital complete achromatopsia. She was followed for 14 years, and her vision and fundus findings were stable. However, the scotopic ERG b-waves at age 22 were smaller than those at age 8, and her FAF images showed increased autofluorescence in both maculae. Genetic examinations revealed combined heterozygous mutations of c.997_998delGA and p.M424V in the CNGA3 gene. The homomeric channel consisting of the CNGA3 subunit with the p.M424V mutation had a weak cGMP-activated current in patch-clamp recordings. In heterologous expression analyses, the expression at the cell surface of the mutant CNGA3 subunit was about 28 % of the wild type. The two novel mutations found in the CNGA3 gene, c.997_998delGA and p.M424V, can cause complete achromatopsia. The vision of the patient was stationary until the third decade of life although the FAF was altered at the age of 22 years.

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.

Lysophosphatidylcholine (LPC) is a bioactive phospholipid that accumulates rapidly in the ischemic myocardium. In recent years, it has been shown that some of the actions of LPC are mediated through the activation of the membrane G proteins. However, the precise mechanism(s) responsible for the LPC-related intracellular signaling in the regulation of cardiac ion channels are still poorly understood. The present study was undertaken to examine whether LPC regulates the slow component of the delayed rectifier K(+) current (I(Ks)) and, if so, what intracellular signals are important for this process. Isolated guinea pig cardiac myocytes were voltage-clamped using the whole-cell configuration of the patch-clamp method. The bath application of 1-palmitoyl-lysophosphatidylcholine (LPC-16) concentration-dependently (EC(50)=0.7μM) and reversibly increased I(Ks) in atrial cells, but failed to potentiate I(Ks) in ventricular myocytes. In contrast, 1-oleoyl-lysophosphatidylcholine (LPC-18:1) only produced a slight I(Ks) increase, and 1-caproyl-lysophosphatidylcholine (LPC-6) or the LPC-16 precursor (phosphatidylcholine) had no effect on I(Ks). Pretreatment of atrial cells with an antibody against the N-terminus of the G2A receptor significantly reduced the LPC-16-induced potentiation of I(Ks). The inhibition of heterotrimeric G protein, phospholipase C (PLC) and protein kinase C (PKC) significantly reduced LPC-16-induced enhancement of I(Ks). Moreover, the blockade of Rho and Rho-kinase by specific inhibitors also inhibited the activity of LPC-16. Immunohistochemical studies demonstrated that G2A was densely distributed in the plasma membrane of atrial myocytes. Therefore, the present study suggests that the activation of a G protein (probably Gα(q)) by LPC-16 potentiates I(Ks) currents through the PLC-PKC and Rho-kinase pathways.

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.

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.R99lambdaH 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)-R99lambdaH 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.

Novel mutations in the L visual pigment gene found in Japanese men with protan color-vision defect having a normal order L/M gene array

Normal visual pigment gene arrays on the human X chromosome have a red gene at the first and a green gene at the second positions. More than half of the arrays have additional green genes downstream, but only the first two genes of the array are likely to be expressed in the retina. An array consisting of four genes in two Japanese participants, A121 and A447, was detected either by pulsed field gel electrophoresis and subsequent Southern hybridization or by single nucleotide primer extension reaction. In both participants, the first gene of the array was green, downstream genes were red and green, and the fourth gene was green. The red gene was determined to be at the second position by comparison of polymorphic sites among the intergenic regions that had been amplified by long-range PCR. Such an array with a reverse normal order of pigment genes, green-red as the first two, has never been reported before. They were expected to have normal color vision but showed protan deficiency (protanomaly), a phenotype lacking the red pigment. The red gene had no mutations in the exons and exon/intron boundaries, but had an A-71C substitution in the promoter in both participants.

The KCNE proteins (KCNE1 through KCNE5) function as beta-subunits of several voltage-gated K(+) channels. Assembly of KCNQ1 K(+) channel alpha-subunits and KCNE1 underlies cardiac I(Ks), while KCNQ1 interacts with all other members of KCNE forming complexes with different properties. Here we investigated synergic actions of KCNE1 and KCNE2 on functional properties of KCNQ1 heterologously expressed in COS7 cells. Patch-clamp recordings from cells expressing KCNQ1 and KCNE1 exhibited the slowly activating current, while co-expression of KCNQ1 with KCNE2 produced a practically time-independent current. When KCNQ1 was co-expressed with both of KCNE1 and KCNE2, the membrane current exhibited a voltage- and time-dependent current whose characteristics differed substantially from those of the KCNQ1/KCNE1 current. The KCNQ1/KCNE1/KCNE2 current had a more depolarized activation voltage, a faster deactivation kinetics, and a less sensitivity to activation by mefenamic acid. These results suggest that KCNE2 can functionally couple to KCNQ1 even in the presence of KCNE1.

Previous study showed noinduction of the reporter gene (-3174/+14) of UGT1A1 in HepG2 by bilirubin, but induction by dexamethasone (DEX). This induction was enhanced seven-fold by the co-expression of human glucocorticoid receptor (GR) and was inhibited by a GR antagonist, RU486, indicating stimulation by DEX-GR. Meanwhile, we could not detect stimulation by beta-estradiol, phenobarbital or rifampicin (RIF) in the presence of GR. We investigated the position playing a role in this induction by GR in the promoter region of UGT1A1 using deletion mutants, and clarified the essential sequence (-75/-63) for the binding site of hepatocyte nuclear factor 1 (HNF1). However, GR did not bind directly to this sequence, because UGT-PE2 did not compete for binding to a glucocorticoid responsive element (GRE) probe in an electrophoretic mobility shift assay (EMSA) method. Labeled [(32)P]DNA probe of HNF1 binds with nuclear extracts as shown by the EMSA. This shift of the complex of probe-protein was not inhibited by unlabeled GRE but was inhibited by unlabeled HNF1 element. This shift was not influenced by the addition of anti-GR, but was super-shifted by the addition of anti-HNF1. GR did not stimulate the induction of HNF1, because we detected no-elevation of the mRNA level of HNF1 by reverse transcription-polymerase chain reaction (RT-PCR). Therefore, the induction of UGT1A1 by DEX-GR did not depend on the elevation of HNF1 but on the interaction of GR with HNF1 or the activation of HNF1 through the transcription of other proteins. Also given the lack of evidence of binding of DEX-GR to HNF1 in the EMSA, the data suggest that the mechanism of DEX-GRE effect on HNF1 is indirect by whatever mechanisms.

TEX28 gene (fTEX) is present immediately downstream of the red/green visual pigment gene array on the human X chromosome. Its pseudogene (pTEX) that lacks exon 1 is present within the array between pigment genes. We found that both fTEX and pTEX genes had a 697 bp insertion/deletion polymorphism in their introns 3. In color-normal male subjects, the frequency of the 697 bp region was 43% (40/94) in pTEX and 97% (91/94) in fTEX in the array of Red-pTEX-Green-fTEX and 10% (9/94) in pTEX and 87% (41/47) in fTEX in the array of Red-pTEX-Green-pTEX-Green-fTEX. These results suggest that normal arrays with multiple green genes may have arisen through gene duplication rather than unequal homologous crossover. In color-vision-deficient male subjects with a single-gene array, the frequency of the 697 bp region was 83% (25/30) in the array of Green-fTEX and 66% (74/112) in the array of Red-fTEX. In color-vision-deficient male subjects with a 2-gene array, the frequency of the region was 44% (16/36) in pTEX and 97% (35/36) in fTEX in the array of Green-pTEX-Green-fTEX and 75% (18/24) in pTEX and 92% (22/24) in fTEX in the array of Red-pTEX-Red-fTEX. These results suggest that 2-green-gene arrays have arisen through unequal homologous crossover between a normal 2-gene array and a single-green-gene array. With data from a long-range PCR method using the insertion/deletion polymorphism, we proposed a structure of the second gene of 3-gene arrays, Green-pTEX-Green-pTEX-Green-fTEX and Red-pTEX-Red-pTEX-Red-fTEX, in color-vision-deficient subjects.

Human UDP-glucuronosyltransferase (UGT) 1A1 is only enzyme in the conjugation of bilirubin for prevention of hyperbilirubinemia and jaundice. Deletion or mutation of the UGT1A1 gene causes Crigler-Najjar syndrome or Gilbert's syndrome. We previously reported the functional promoter region for expression of UGT1A1 [Hepatology Research 9, 152–163 (1997)]. We investigated the influence of some drugs on the transient transfection assay of the luciferase reporter gene containing the 5′-promoter region −3174/+14 of UGT1A1 in HepG2 cells. Among drugs investigated, dexamethasone was the most effective at the range of concentration of 10–100 μM, whereas stimulation by β-estradiol was not found. We also could not find stimulation by bilirubin of the endogenous main substrate for UGT1A1. Stimulation by dexamethasone was continued for 48 hr. The luciferase reporter gene containing the 5′-region of −97/+14 was induced by dexamethasone but the gene of the 5′-region −53/+14 was not. The region −97/−53 is essential for induction by dexamethasone. This region contains HNF1 element, therefore, we speculated that dexamethasone directly and/or indirectly stimulates UGT1A1 expression through this HNF1 region in the promoter region of UGT1A1. Thus, we clarified that UGT1A1 was induced by dexamethasone and the key position was the region (−97/−53) in UGT1A1 promoter. Abbreviations: UGT – UDP-glucuronosyltransferase; PB –phenobarbital; PBREM – PB response enhancer module; Dex –dexamethasone; GR – glucocorticoid receptor; GRE – glucocorticoidresponsive element; CAR – constitutive androstane receptor; PXR –pregnenolone xenobiotic receptor; HNF – hepatocyte nuclear factor.

The L-cone/M-cone visual pigment gene arrays were analyzed in a group of 63 Japanese females consisting of 7 applicants for examination of their carrier status, 14 color-deficient females, 6 obligate carriers with no genotypic data available for affected father or sons, and 36 color-normals. The first and the downstream genes, the entire region from the promoter to exon 6, were each amplified very efficiently by the long-range PCR to give products of 15.8 and 14.4 kb, respectively. The products were gel-purified and used as the template in the second PCR for exon 5. The region from intron 4 of the last genes, to the nearest neighbor gene, TEX28, was also efficiently amplified by the long-range PCR and the gel-purified products (27.5 kb) were used as the template in the second PCR for exon 5. The status of the 7 applicants was thought to be 3 non-carriers, 2 protan carriers and 2 deutan carriers. All of the 14 color-deficient females had unusual arrays in which an M gene was present as the first gene, an L gene(s) was present downstream, or a single L gene constituted both of the two arrays. One protanopic subject, A348, had an L gene as one of the first genes. The 6 obligate carriers also had unusual arrays with the exception of the mother of the A187, a male subject with pigment color defect. In the 36 color-normal individuals, 4 had downstream L genes. The long-range PCR method is useful for analysis of the L/M visual pigment genes.

In 524 Japanese individuals with deutan colour vision defect, 76 had a normal-order pigment gene array, where the L gene is at the first position and the M gene(s) is located downstream. Of these 76 individuals, 69 had a -71A>C substitution in the M gene without any other mutation. Because the expression of L/M genes is up-regulated by thyroid hormone (T3) in human retinoblastoma WERI cells, we examined the effects of T3 on promoter activity; T3 increased the activity of the -71A promoter 2-fold, but it had no effect on the -71C promoter. Similarly, the -71C promoter was much less activated by T3 than the -71A promoter in HEK293 cells expressing thyroid hormone receptor isoform β2. Such a weak response of the -71C promoter to T3 may cause a decrease in the number of M cones and/or the density of M pigment during the differentiation of M cones. The average Rayleigh match midpoint was 18.9 ± 4.1 in 162 ordinary deuteranomaly individuals, but was 37.3 ± 9.1 in 63 deuteranomaly individuals with -71C. The -71A>C substitution was found to be specific to eastern Asia. These results suggest that there may be a new subset of deuteranomaly associated with -71C in the Japanese (and probably eastern Asian) population(s).

Background. There have been many studies on anti-lipopolysaccharide (LPS) agents and LPS-neutralizing agents; however, there have been no reports on the changes in clinical status and mediators that occur when these agents are used. Polymyxin (PMX) (treatment using a column containing polymyxin B-immobilized fiber) removed circulating endotoxin, and reduced various cytokines within 120 min, even in patients with high levels of plasma cytokines. Our purpose was examine the mechanisms of PMX treatment by which plasma cytokines are reduced by endotoxin neutralization with polymyxin B, even during therapy for sepsis and/or endotoxin shock. Methods. We studied the interaction between nuclear factor κB (NF-κB) binding activity and tumor necrosis factor α (TNF-α) secretion in an experimental system using LPS-stimulated human peripheral blood mononuclear cells (PBMCs), after neutralization of LPS with polymyxin B. PBMCs were incubated with LPS in vitro, and TNF-α secretion and NF-κB activation were assessed. We then studied the changes in NF-κB activation and TNF-α secretion when both polymyxin B and LPS were added simultaneously and when polymyxin B was added after 30 or 120 min of incubation with LPS. Results. Immediate inhibition of NF-κB binding activity and suppression of TNF-α secretion were observed after LPS neutralization with polymyxin B regardless of whether PBMCs were already producing TNF-α. Conclusions. These findings may indicate one of the mechanisms operating in the clinical changes that occur after circulating endotoxin removal, and are likely to have therapeutic value, even for patients with high proinflammatory cytokine levels.

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.

Papillary serous carcinoma of the peritoneum (PSCP) is a primary neoplasm of peritoneal origin, and is histologically difficult to differentiate from papillary serous carcinoma of the ovary (PSCO). PSCP is frequently accompanied by many peritoneal tumors, and has been managed as a disseminated disease. In previous reports, however, the clonality of the tumors has not been fully discussed. Recently, the significant roles of the p53 and BRCA1 genes in PSCP have been reported. In this study, we investigated immunohistochemical staining for p53 proteins, and investigated p53 gene mutations, using DNA sequencing analysis, to clarify the clonality of PSCP tumors. Immunohistochemically, all the tumor samples demonstrated nuclear overexpression of p53 proteins, and the DNA sequencing analysis of the p53 gene showed diverse point mutations at codons 167 and 192 in two of four anatomically different tumors. In conclusion, the possibility of polyclonality of PSCP tumors is suggested.

Conflicting results have been obtained in previous studies concerning the adaptation of intestinal blush border membrane enzymes to starvation. This study was designed to clarity the changes in these enzymes under starvation conditions, using a molecular biological approach. Sprague-Dawley rats were starved or given total parenteral nutrition (TPN) for 5 days. Rats allowed free access to food were used as controls. Changes in the activity and expression of jejunal brush border membrane enzymes were compared between three groups. In the starved group, aminopeptidase N and dipeptidyl peptidase IV activity was significantly elevated to 177% and 166%, respectively, of control values. In contrast, sucrase and maltase activity was significantly decreased. The activity of these peptidases also tended to be increased at the renal brush border membrane. Up-regulation of peptidase activity was not evident in the TPN group. Western and Northern blot analysis revealed that the changes in aminopeptidase N activity were attributable to increases in the protein and mRNA level. The activity and expression of brush border membrane peptidases in rat jejunum is up-regulated during starvation, and these changes are considered to be an effect of whole-body malnourishment, rather than an absence of luminal nutrition.

Congenital color-vision deficiencies are frequent among males, 4.7-8.0%, suggesting that female carriers are present at a frequency of 9-15%. The purpose of this study was to determine whether carriers could be detected by analysis of the visual pigment genes.DNA from 29 males with congenital color-vision deficiencies, from their mothers, and from 117 randomly-selected females was analyzed. The most upstream genes, the downstream genes, and the most downstream genes in the red/green pigment gene arrays were amplified separately by PCR. Exon 5 of each gene was analyzed by single-strand conformation polymorphisms (SSCP).Analysis of the visual pigment genes suggests that one of the 29 mothers examined is a female protan and two others are carriers of both protan and deutan defects. The remaining 26 mothers were confirmed to be carriers of congenital color-vision deficiencies. Unusual patterns were observed in 15 (13%) of the randomly-selected females; among them, 5 appeared to be protan carriers and at least 4 to be deutan carriers.Female carriers of congenital color-vision deficiencies can be detected by analysis of the visual pigment genes. Since the proportion of females showing unusual patterns was slightly higher than expected, some must be false-positives and require more detailed examination.

Recent advancements in molecular biology have revealed genetic aspects of congenital color vision deficiencies (CVD). In many cases of CVD, the genotypes and phenotypes coincide with each other. But it is also known that there are cases in which the genotype alone cannot explain the clinical diagnosis of CVD. So it is still unclear whether routine gene analysis is useful as a clinical diagnostic tool for this anomaly. We evaluated the clinical usefulness of genetic analysis for congenital red-green color-vision deficiencies. The nucleotide sequences of red-green pigment genes of 62 CVD cases and 113 color-normal males were determined using PCR and a DNA sequencer (ABI PRISM 310 Genetic Analyzer). The CVD cases were diagnosed using clinical color-vision tests including an anomaloscope. The color-normal subjects were tested with Ishihara plates and, if needed, with an anomaloscope. All normals had two types of exon 5, i.e., both red and green pigment genes. Forty-seven (76%) of 62 CVD cases had genotypes that coincided with their phenotypes. Three dichromats (one protanope and two deuteranopes) had genetic potential to be anomalous trichromats. Twelve deutans (one deuteranope and 11 deuteranomals) could not be differentiated from the normals: they had both red and green types of genes, as did the normals. All in all, 15 cases (24%) had some inconsistencies between the phenotype and the genotype. Pigment gene analysis alone could not fully differentiate CVD from normals. © 2000 John Wiley & Sons, Inc. Col Res Appl, 26, S89–S92, 2001

Deuteranomaly, and to a lesser extent deuteranopia, are associated with a 5′ green-red 3′ (G-R) visual pigment hybrid gene. Such G-R hybrid genes may also exist in males with normal color vision. This discrepancy can be explained if the position of the G-R hybrid gene in the array determines the probability of its expression in the retina. In accord with our previous findings, we confirm that when a G-R hybrid gene occupies the second position in the array, a deutan defect results (N = 2); whereas when a normal green pigment gene does, normal color vision ensues (N = 3). Regarding gene expression as a function of position in the array, in 5 postmortem retinae from individuals whose arrays contained one red and two green pigment genes that differed at a polymorphic site in exon 5, only the green pigment gene that occupied the second position in the array was expressed into retinal mRNA. In conclusion, our data suggest that only the first two genes in the X-chromosome linked visual gene array are expressed in the retina and, therefore, influence the color vision phenotype. Green-red hybrid genes cause deuteranomaly only if they occupy the second position in the array. © 2000 John Wiley & Sons, Inc. Col Res Appl, 26, S79–S83, 2001

Alanyl aminopeptidase (AAP) was purified to homogeneity from human seminal plasma. The calculated molecular weight of the purified enzyme was approximately 137,000+/-5,000 from light scattering, 140,000 (main) and 137,000 (minor) from non-denatured PAGE and 153,000 from SDS-PAGE in the absence or presence of 2-mercaptoethanol (2-ME). These findings suggest that the enzyme is monomeric in form in human seminal plasma. The enzyme hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order of Kcat/Km values of AAP at optimal pH (pH 7.5) was Ala- > Lys-Ala- > or = Met- > Leu- > Phe- > Arg- > or = Arg-Arg- > Lys- > Gly-MCAs. AAP was potently inhibited by bestatin, leuhistin, actinonin, amastatin, and 1,10-phenanthroline. These findings suggest that AAP is an aminopeptidase. We determined that the amino acid sequence of the first 22 residues of the enzyme was Ser1-Thr-Thr-Pro-Ser5-Ala-Ser-Ala-Thr-Thr10-Asn-Pro-Al a-Ser-Ala15-Thr-Thr-Leu-Asp-Gln20-Ser-Lys-. This sequence was completely coincident with that downstream of the transmembrane site of human intestinal alanyl aminopeptidase N (CD13). We also isolated cDNA encoding AAP from human prostate cDNA library, sequenced its structure, and confirmed human seminal plasma AAP to be identical with alanyl aminopeptidase N. We postulated that native human seminal plasma alanyl aminopeptidase is released into the seminal plasma after the specific site is cleaved by elastase or an elastase-like enzyme. The enzyme level in human seminal plasma determined by single radial immunodiffusion was 5.2+/-3.2 mg/100 ml (mean+/-SD, n=20) in individuals 20-47 years of age. AAP was immunohistochemically stained in the luminal site-cell membrane of epithelial cells in the prostatic gland and ductuli efferentes of the testis.

Human beta- and gamma-cytoplasmic actin genes (ACTB and ACTG1) were mapped to chromosomes 7p22 and 17q25, respectively, by fluorescence in situ hybridization (FISH). Four processed pseudogenes, beta-actin-related ACTBP9 and gamma-actin-related ACTGP1, ACTGP3, and ACTGP9, were isolated from human libraries. By PCR of somatic cell hybrid DNAs, ACTBP9 and two beta-actin-related pseudogenes (ACTBP7 and ACTBP8) were mapped to human chromosomes 18, 15, and 6, respectively. The gamma-actin-related pseudogenes were mapped by FISH to chromosomes 3q23 (ACTGP1), 20p13 (ACTGP3), and 6p21.1 (ACTGP9).

beta-Microseminoprotein was very efficiently purified from human seminal plasma with only three steps including DEAE-Sephacel and Zinc-chelate Sepharose CL-6B column chromatography. The purified protein was a non-glycoprotein with a molecular weight (M(r)) of 19,000 and 17,000 on gel filtration and reduced SDS-PAGE, respectively. The protein gave six bands from M(r) 15,600 to 25,500 on non-reduced SDS-PAGE. The characterization including the molecular weight, amino acid sequence of N-terminus and concentrations in various body fluids is discussed. Furthermore, the immunohistochemical localization of the protein among various human tissues is demonstrated.

Human genes for cardiac (ACTC), skeletal (ACTA), and vascular type (aortic type or α-) smooth (ACTSA) muscle actins have been localized to chromosomes 15q14, 1q42.1, and 10q23.3, respectively, by fluorescencein situ hybridization.

The human gene for smooth muscle actin (enteric type, ACTA3) has been isolated, and three overlapping clones, lambda HACTSG-17, -2, and -112, were used as probes for fluorescence in situ hybridization of human chromosomes. The gene was localized to chromosome 2p13.1. To clarify the molecular nature of the HindIII RFLP present in the first intron of the gene, the 1105-bp EcoRI-BamHI fragment contained in lambda HACTSG-17 was sequenced. PCR with primers designed from the determined sequence yielded either the 463- or the 439-bp product or both, using human DNA as template. The 463-bp product was cleavable with HindIII, but the 439-bp product was not. Comparison of their nucleotide sequences revealed that they differ in the presence/absence of a 24-bp sequence harboring a HindIII restriction site. Therefore, analysis of PCR products by size has been shown to be sufficient to detect the RFLP. The allelic frequency on 156 chromosomes was determined by PCR to be 45 (439 bp, corresponding to the formerly designated A1 allele):55 (463 bp, A2 allele) in the Japanese population.

The gene for human beta-microseminoprotein (MSP, beta-inhibin, prostatic secretory protein94, or PSP94) was isolated. The nucleotide sequence of its upstream region (2,860 bp), exon 1 (35 bp), and a part of the following intron (218 bp) was determined. Transient transfection analysis on MSP-expressing (PC-3) and -nonexpressing (HepG2) cells using luciferase reporter plasmids suggested that the region from -2738 to -276 is not essential for the basal promoter activity, and that the regions from -275 to -207 and from -186 to -128 function in a cell-specific manner. The chromosome locus of the gene (MSMB) was determined through application of PCR to the DNAs of rodent-human somatic cell hybrids and also by the fluorescence in situ hybridization technique to be region q11.2 of chromosome 10.

Rat and mouse cDNAs for Zn-alpha 2-glycoprotein (Zn alpha 2gp) were isolated from liver libraries (lambda gt11) and compared with the human one. The lengths of cDNA inserts analyzed were 1,233 and 1,273 nucleotides for rat and mouse, respectively. The deduced amino acid sequences suggested that rat and mouse Zn alpha 2gp proteins consist of 279 and 290 amino acid residues in the mature form, respectively. They have 59.4% (rat) and 58.6% (mouse) identities in amino acid sequence with the human counterpart, and between rat and mouse the identity is 88.5%. Among the three domains, domain B is best conserved; the identities are 74.7, 73.6, and 95.6% between human and rat, human and mouse, and rat and mouse, respectively. Four cysteine and eight tryptophan residues are all conserved, and two of the three asparagine residues that carry a glycan in the human protein are conserved. Analysis of rat tissues by Northern blot suggested that its mRNA is expressed in liver, and, to a much lesser extent in submandibular gland, lung, kidney, and stomach. A more detailed study by in situ hybridization demonstrated that some epithelial cells of renal tubules and the isthmus and the neck zone cells of gastric fundic glands express Zn alpha 2gp mRNA.

Among the 447 Japanese men with deutan color-vision deficiency that we analyzed, 61 had a normal order array of L/M pigment genes. Three of the 61 men had an exonic mutation, but the other 58 had no mutations even in the flanking introns of their M genes. In these 58 men, 55 had a -71A --> C substitution in the M gene. Two hypotheses were built up for the substitution: it is in linkage disequilibrium with a genuine cause of deficiency in the introns, or itself is the cause of the deficiency. For the first hypothesis, we sequenced entire regions of both the L and M genes in 30 color-normal Japanese men who had one each of the L and M genes to understand normal variations of the introns. Fifty-two already known and 15 newly identified polymorphic sites could be classified into three categories: those with no polymorphisms in the Japanese group, those essentially different between the L and the M genes, and the others. We then sequenced the entire region of the M genes in 12 representative deutan individuals with a normal gene-order array but found no significant mutations. For the second hypothesis, we performed a reporter assay and found that the M gene promoter with -71C had a 60-70% reduction in activity when compared to that with -71A. These results suggest that the -71A --> C substitution is not in linkage disequilibrium with an intronic mutation, but the substitution itself may affect the transcription of the M gene, leading to deutan deficiency.

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.

Licochalcone A, 3-a,a-dimethylallyl-4,4'-dihydroxy-6-methoxychalcone, from the root of Glycyrrhiza inflata Beta (Leguminosae) (Xin-jiang liquorice) showed anti-inflammatory action towards mouse ear edema induced by arachidonic acid (AA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) by topical application. Anti-tumour promoting action of licochalcone A was also observed in vivo for mouse skin papilloma initiated by dimethylbenz[a]anthracene (DMBA) and promoted by TPA. It inhibited in vitro 32Pi-incorporation to phospholipids in HeLa cells promoted by TPA. A competitive interaction of licochalcone A with the TPA-receptors in the cell membrane has been suggested.

The neural crest gives rise to a variety of tissues, including peripheral neurons, Schwann cells, melanocytes and ectomesenchymal cells, which include the smooth-muscle cells of large arteries. Cell lines derived from neuroblastoma (a neural-crest tumor) exhibit at least 2 distinct morphological cell types, a neuroblastic phenotype (N-type) and an epithelial-like phenotype (S-type) with characteristics of substrate-adhesiveness. We have analyzed 17 human neuroblastoma cell lines using a panel of monoclonal antibodies (MAbs) against cytoskeletal proteins. Three neuroblastoma cell lines (KP-N-SI, KP-N-YN and SMS-KCN) bound an alpha-smooth-muscle actin antibody. In addition, one of these lines (KP-N-SI) bound anti-desmin MAbs as determined by indirect immunofluorescence. A total of 8 cloned cell lines were obtained from the above parent cell lines. These were composed of either N- or S-type cells and were confirmed to have the same neuroblastoma origin as each parent cell line by chromosomal analysis. Alpha-smooth-muscle actin and desmin were demonstrated in the S-type cloned cells by indirect immunofluorescence, as well as by 2-dimensional Western blot analysis. These results were confirmed by Northern blot analysis using a specific probe (pSH alpha SMA-3' UT) to human alpha-smooth-muscle actin mRNA. These ascertain the presence of alpha-smooth-muscle actin and desmin in neuroblastoma cell lines. These data show that, in addition to giving rise to cells with neural, Schwann-cell and melanocyte markers, neuroblastoma can also give rise to the cells expressing smooth-muscle cell markers.

Human 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.

Since Pd-II [(+)anomalin, (+)praeruptorin B], a seselin-type coumarin, was found to inhibit tumor promoter induced phenomenon in vitro, the effect of Pd-II on the in vivo tumor-promoting action of 12-O-tetradecanoylphorbol-13-acetate (TPA) in 7,12-dimethylbenz[a]anthracene-initiated mouse skin was investigated. Pd-II, applied 40 min before the TPA treatment, at a dose of 10 mumol/painting, completely suppressed tumor formation up to 20 weeks of tumor promotion, without any toxicity. Besides Pd-II, various anti-tumor-promoter coumarins were found in the traditional Chinese medicine Qian-Hu, from which Pd-II was obtained. These coumarins may be useful for the development of an effective method to prevent cancer.

The changes of protein kinases (PKs) in the nuclear matrix of HL-60 cells during the differentiation by retinoic acid (RA) were examined by in situ assay after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the nuclear matrix of HL-60, at least seven species of PKs (83, 63, 58, 46, 42, 38, and 31 kDa) were always detected. Among these PKs, 63 kDa PK was increased dramatically during differentiation by RA. Two-dimensional electrophoresis revealed that two species of 63 kDa PKs (pI 5.0 and 5.4) were presented in the nuclear matrix and increased similarly during the differentiation. This increase of 63 kDa PKs may be related to the lobulation of the nucleus and the nuclear matrix accompanying the differentiation by RA.

Thirty-nine missense mutations, which had been identified in rod monochromacy or related disorders, in the CNGA3 subunit of cone photoreceptor cGMP-gated channels were analyzed. HEK293 cells were transfected with cDNA of the human CNGA3 subunit harboring each of these mutations in an expression vector. Patch-clamp recordings demonstrated that 32 of the 39 mutants did not show cGMP-activated current, suggesting that these 32 mutations cause a loss of function of the channels. From the remaining 7 mutants that showed cGMP-activated current, two mutations in the cyclic nucleotide-binding domain, T565M or E593K, were further studied. The half-maximal activating concentration (K(1/2)) for cGMP in the homomeric CNGA3-T565M channels (160microM) was 17.8-fold higher than that of the homomeric wild-type CNGA3 channels (9.0microM). Conversely, the K(1/2) for cGMP in the homomeric CNGA3-E593K channels (3.0microM) was 3-fold lower than that of the homomeric wild-type CNGA3 channels. These results suggest that the T565M and E593K mutations alter the apparent affinity for cGMP of the channels to cause cone dysfunction, resulting in rod monochromacy.

The L-cone/M-cone visual pigment gene arrays were analyzed in 125 Japanese males with protan color-vision deficiency. Arrays were successfully determined in 62/65 subjects with protanopia and 57/60 protanomaly subjects. Among the 62 protanopia subjects, 48 (77%) had an array consisting of a single 5′ L–M hybrid gene (PS-array) or a 5′ L–M hybrid gene followed by an M gene(s) that was structurally identical to the hybrid gene (PI-array). In the remaining 14 subjects, 11 had an array consisting of a 5′ L–M hybrid gene followed by an M gene(s) that was structurally different from the hybrid gene (PD-array) and 3 subjects had an apparently normal array consisting of a single L gene followed by an M gene(s) (PN-array). In the 11 subjects with the PD-array, subject A67 had an 11 bp-deletion in exon 3 of the downstream genes and 6 had an A-71C substitution in the second gene of the array. In the 3 subjects with the PN-array, subject A289 had a missense mutation (Pro231Leu) in exon 4 of the L gene. When the function of the missense mutation was studied by in vitro reconstitution of visual pigments, it was found to be deleterious to both cone opsin and rhodopsin. Among the 57 protanomaly subjects, 49 (86%) had the PD-array, but 25 subjects had a difference only in exon 2 between the first and downstream genes that suggested a contribution of exon 2-encoded difference in the M pigment to color-discrimination. In the remaining 8 subjects, 2 had the PS-array, 2 had the PI-array and the other 4, including subject A89 with a missense mutation (Glu338Gly) in the L gene, had the PN-array. Genotype–phenotype relationships in protan color-vision deficiency are discussed.

We studied 247 Japanese males with congenital deutan color-vision deficiency and found that 37 subjects (15.0%) had a normal genotype of a single red gene followed by a green gene(s). Two of them had missense mutations in the green gene(s), but the other 35 subjects had no mutations in either the exons or their flanking introns. However, 32 of the 35 subjects, including all 8 subjects with pigment-color defect, a special category of deuteranomaly, had a nucleotide substitution, A−71C, in the promoter of a green gene at the second position in the red/green visual-pigment gene array. Although the −71C substitution was also present in color-normal Japanese males at a frequency of 24.3%, it was never at the second position but always found further downstream. The substitution was found in 19.4% of Chinese males and 7.7% of Thai males but rarely in Caucasians or African Americans. These results suggest that the A−71C substitution in the green gene at the second position is closely associated with deutan color-vision deficiency. In Japanese and presumably other Asian populations further downstream genes with −71C comprise a reservoir of the visual-pigment genes that cause deutan color-vision deficiency by unequal crossing over between the intergenic regions.

The DNAs from 217 Japanese males with congenital red/green color-vision deficiencies were analyzed. Twenty-three subjects had the normal genotype of a single red gene, followed by a green gene. Four of the 23 were from the 69 protan subject group and 19 of the 23 were from the 148 deutan subject group. Three of the 23 subjects had missense mutations. The mutation Asn94Lys (AAC--AAA) occurred in the single green gene of a deutan subject (A155). The Arg330Gln (CGA--CAA) mutation was detected in both green genes of another deutan subject (A164). The Gly338Glu (GGG--GAG) mutation occurred in the single red gene of a protan subject (A89). Both normal and mutant opsins were expressed in cultured COS-7 cells and visual pigments were regenerated with 11-cis-retinal. The normal red and green opsins showed absorbance spectra with lambda(max) of 560 and 530 nm, respectively, but the three mutant opsins had altered spectra. The mutations in Asn94Lys and Gly338Glu resulted in no absorbance and the Arg330Gln mutation gave a low absorbance spectrum with a lambda(max) of 530 nm. Therefore these three mutant opsins are likely to be affected in the folding process, resulting in a loss of function as a visual pigment.

We found that human kinin-free high-molecular-weight kininogen (kf-HK) significantly inhibited vitronectin-mediated migration (haptotaxis) and invasive potentiation (haptoinvasion) of osteosarcoma (MG-63) cells but that HK, LK, the common heavy chain of HK and LK, and the light chain (D6(H)) of HK had no inhibitory effect. Recombinant GST-D5(H) (histidine-rich region of HK) obtained from Escherichia coli. (BL21) also inhibited both haptotaxis and haptoinvasion to about 30% of the control level in a dose-dependent manner. These findings suggest that a specific region of D5(H) is responsible for the inhibition of cell haptotaxis and haptoinvasion. Among the seven synthetic peptides covering D5(H), peptide H(479)KHGHGHGKHKNKGK(493) (P-5) inhibited both haptotaxis and haptoinvasion in a dose-dependent manner, suggesting that P-5 could possibly be utilized to prevent primary and secondary metastases of tumor cells.

Thiamin pyrophosphokinase (EC 2.7.6.2) catalyzes the pyrophosphorylation of thiamin with adenosine 5′-triphosphate to form thiamin pyrophosphate. A mouse thiamin pyrophosphokinase cDNA clone (mTPK1) was isolated using a combination of mouse expressed sequence tag database analysis, a two-step polymerase chain reaction procedure, and functional complementation screening with aSaccharomyces cerevisiae thiamin pyrophosphokinase-deficient mutant (thi80). The predicted protein contained 243 amino acid residues with a calculated molecular weight of 27,068. When the intact mTPK1 open reading frame was expressed as a glutathione S-transferase fusion protein in Escherichia coli lacking thiamin pyrophosphokinase, marked enzyme activity was detected in the bacterial cells. The corresponding 2.5-kilobase pair mRNA was expressed in a tissue-dependent manner and was found at relatively high levels in the kidney and liver, indicating that the mode of expression of mTPK1 genes differs with cell type. The expression ofmTPK1 genes in cultured mouse neuroblastoma and normal liver cells was unaffected by the thiamin concentration in the medium (10 μm versus 3.0 nm). This is the first report on identification of the primary sequence for mammalian thiamin pyrophosphokinase.

Objective To investigate the gene expression of estrogen receptor (ER) α and ERβ in human articular chondrocytes. Methods 16 articular cartilage specimens were obtained from 15 patients during surgery. Three of the specimens were from men and 13 from women; three from hip joints and 13 from knee joints; four were normal and 12 showed osteoarthritic cartilage. Total RNA was extracted from the articular chondrocytes and the expression of both ERα and ERβ genes was investigated by the reverse transcription-polymerase chain reaction (RT-PCR) method. Results Gene expressions of ERα were detected in all specimens and those of ERβ were found in 15 specimens by the RT-PCR method. There was a significant correlation between the amounts of ERα and ERβ. Expression levels of both genes were significantly higher in men than in women. There were no significant differences in the expression levels of both ER genes between the hip and knee joint sites, nor between normal and osteoarthritic tissues. Conclusion This study is to our knowledge the first to demonstrate the gene expression of both ERα and ERβ in human articular chondrocytes. Since there are some functional differences between the two receptors, the effects of estrogen on cartilage metabolism should be elucidated by two different receptor mechanisms.{copy}

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.

The promoter of the human gene for beta-microseminoprotein (MSP) was analyzed by transient transfection assay using LNCaP cells. The region between -2738 and -207 was not essential for the basal promoter activity, but regions from -206, -108, and -83 were necessary. Forskolin induced promoter activity to 3- to 14-fold when constructions containing the possible cAMP response element (CRE, -60 to -53) were used, but did not do so when it was deleted from them. In a gel-mobility shift assay using a probe corresponding to -68 to -48, the shifted band was competed with a consensus CRE, and supershifted when the anti-CREB-1 antibody was included. Forskolin added to LNCaP cells increased MSP mRNA to 6-fold within 12 h. These results suggest that the expression of MSP gene is regulated by cAMP via the CRE present in the promoter. Hormones such as (-)-epinephrine or vasoactive intestinal polypeptide also increased MSP mRNA to 2- to 3-fold.

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.