Non-synonymous single nucleotide alterations in the microsomal epoxide hydrolase gene and their functional effects.

1. By sequencing genomic DNA from 72 established cell lines derived from Japanese individuals, we detected 25 single nucleotide alterations in the microsomal epoxide hydrolase (EPHX1) gene. Of them, five were exonic alterations resulting in amino acid alterations (77C>G, T26S; 128G>C, R43T; 337T>C, Y113H; 416A>G, H139R; 823A>G, T275A). The T26S, R43T, Y113H and H139R substitutions were found at relatively high frequencies and seemed to be polymorphic, and T26S and T275A were novel. 2. To examine the effects of these amino acid alterations on EPHX1 function, EPHX1 cDNA constructs of wild-type and five variants were transfected into COS-1 cells, and their hydrolytic activities for cis-stilbene oxide were determined in vitro. Although all of the transfectants expressed EPHX1 mRNA and protein at similar levels, the variant H139R protein was expressed at a significantly higher level (128% of the wild-type). K(m) values were not significantly different between the wild-type and variants. 3. Increase (140%) in the enzymatic activity (V(max)) of the variant H139R was accompanied by the increased EPHX1 protein level without any significant change in the intrinsic EPHX1 activity. On the other hand, the variant R43T showed increased values for V(max) and clearance (V(max)/K(m)) (around 130%) both on a microsomal protein basis and on a EPHX1 protein basis. 4. These results suggest that R43T as well as H139R increase epoxide hydrolase activity.

Measurement of plasma brain natriuretic peptide level as a guide for cardiac overload.

OBJECTIVES: We examined whether measurement of the plasma BNP concentrations might be useful for the early diagnosis of the existence and severity of disease in patients with heart disease in daily clinical practice. METHODS AND RESULTS: The plasma BNP and ANP concentrations in 415 patients with heart disease and hypertension and 65 control subjects were measured. Patients with heart disease had higher plasma BNP and ANP concentrations than did those with hypertension or control subjects. Among the etiology of cardiac diseases, specifically dilated cardiomyopathy and hypertrophic cardiomyopathy, was associated with the highest plasma BNP concentrations, whereas dilated cardiomyopathy was associated with the highest plasma ANP concentrations. Plasma BNP concentrations showed an increase as the severity of the heart disease, as graded according to the NYHA classification of cardiac function, increased. In both patients with heart disease and hypertension, the plasma BNP values were higher in those who had abnormalities in their echocardiogram and electrocardiogram as compared to those without any abnormalities. The plasma BNP levels also showed a significant correlation with left ventricular wall thickness and left ventricular mass. On the other hand, the plasma ANP levels showed significant correlations with left ventricular dimension. Receiver operative characteristic analysis revealed that plasma BNP levels showed substantially high sensitivity and specificity to detect the existence of heart diseases. CONCLUSION: Measurements of the plasma BNP concentrations is useful to detect the existence of the diseases, and abnormalities of left ventricular function and hypertrophy in patients with heart disease in daily clinical practice.

Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1.

Heme controls expression of genes involved in the synthesis of globins and heme. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero-oligomers with the small Maf family proteins. We show here that heme binds specifically to Bach1 and regulates its DNA-binding activity. Deletion studies demonstrated that a heme-binding region of Bach1 is confined within its C-terminal region that possesses four dipeptide cysteine-proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA-binding activity of Bach1 as a MafK hetero-oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased levels of heme inactivate the repressor Bach1, resulting in induction of a host of genes with MARES:

Non-synonymous single nucleotide alterations found in the CYP2C8 gene result in reduced in vitro paclitaxel metabolism.

By sequencing genomic DNA from 73 established cell lines derived from Japanese individuals, we detected 9 single nucleotide polymorphisms (SNPs) in the CYP2C8 gene. Of them, 3 exonic SNPs resulted in amino acid alterations (g416a, R139K; a1196g, K399R; c1210g, P404A). The first two alterations were detected concurrently in one cell line and thought to be the same as CYP2C8*3. To examine the effects of these amino acid alterations on CYP2C8 function, wild-type and four types of variant CYP2C8 cDNA constructs (R139K, K399R, R139K/K399R and P404A) were transfected into Hep G2 cells and their paclitaxel 6a-hydroxylase activities were determined in vitro. Km values were not significantly different from that of the wild-type in any of the variants studied. The variant R139K/K399R showed reduced values for Vmax and clearance (Vmax/Km) similar to those of its single variant, R139K. The variant P404A also showed a significantly lowered clearance due to reduced level of protein expression. These results suggest that not only the double variant (R139K/K399R, CYP2C8*3) but also our novel variant P404A in the CYP2C8 gene are less efficient in paclitaxel metabolism.

Massive deep vein thrombosis after cesarean section treated with a temporary inferior vena cava filter: a case report.

A 25-year-old woman suffered a massive deep vein thrombus in her left common iliac vein extending to the inferior vena cava after an abdominal cesarean section. The massive and floating inferior vena cava thrombus was considered to pose a high risk of pulmonary thromboembolism. After placement of a temporary inferior vena cava filter via the left brachial vein, thrombolytic therapy and anticoagulation therapy were instituted. The filter successfully prevented pulmonary thromboembolism during thrombolytic therapy. This patient was confined to bed because the filter moved vertically with left shoulder joint abduction. Although a temporary inferior vena cava filter is very useful for the prevention of pulmonary thromboembolism in a patient with deep vein thrombus, the appropriate range of activity for such a patient needs careful consideration.

Animal models for X-linked sideroblastic anemia.

Erythroid 5-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells. Several lines of evidence suggest that the expression of ALAS-E is important for the process of erythroid differentiation, which requires a large amount of heme for hemoglobin production. Mutation of human ALAS-E causes the disorder X-linked sideroblastic anemia (XLSA). More than 25 unrelated ALAS-E mutations in XLSA patients have been reported. Most XLSA cases are of the pyridoxine-responsive type, but molecular diagnosis of 1 pyridoxine-refractory type XLSA has also been reported. To examine the roles heme plays during hematopoiesis and to create animal models of XLSA, we disrupted the mouse ALAS-E gene. A chemically induced zebrafish mutant (sau) that lacks ALAS-E has also been isolated. Analysis of these ALAS-E mutants unequivocally demonstrated that ALAS-E is the principal isozyme contributing to erythroid heme biosynthesis In ALAS-E-null mutant mouse embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. This accumulation of iron was in contrast to that in XLSA patients, as typical ring sideroblasts accumulated iron primarily in mitochondria. These observations suggest that the mode of iron accumulation caused by the lack of ALAS-E is different in primitive and definitive erythroid cells. Thus ALAS-E, and hence heme supply, is necessary for erythroid cell differentiation and iron metabolism.

Developmental expression of opioid-binding cell adhesion molecule (OBCAM) in rat brain.

Opioid-binding cell adhesion molecule (OBCAM), a neuron-specific protein, consists of three immunoglobulin (Ig)-like domains anchored to the membrane through a glycosylphosphatidylinositol (GPI)-tail. OBCAM has been presumed to play a role as a cell adhesion/recognition molecule, but its function has not been fully elucidated. We investigated the developmental expression of OBCAM in rat brain by using a monoclonal anti-OBCAM peptide antibody (OBC53). OBCAM was clearly detectable on embryonic day 16 (E16) as assessed by immunoblotting. The expression level increased by the second postnatal week and was maintained at a constant level until week 17. During the early developmental period OBCAM was found to be expressed on postmitotic neurons and to be strongly expressed in at the fiber tracts containing expanding axons, in contrast to the adult brain, in which OBCAM is principally expressed in the gray matter. These findings suggest that the function of OBCAM involves axonal outgrowth.

Effect of the calcium antagonist benidipine hydrochloride on 24-h ambulatory blood pressure in patients with mild to moderate hypertension in a double-blind study against placebo.

The effects of benidipine hydrochloride (benidipine, CAS 91559-74-5), a dihydropyridine calcium antagonist, on the 24-h ambulatory blood pressure were studied by a double-blind test against placebo in 8 patients with essential hypertension. The mean resting systolic (SBP) and diastolic (DBP) blood pressures were 173 mmHg and 104 mmHg, respectively, at the time of patients enrollment. Blood pressure was measured every 30 min for 24 h after a single oral administration of either benidipine (4 mg/day) or the placebo. The mean through/peak (T/P) ratios were calculated from blood pressure measurements obtained using 2-h moving averages, and the smoothness index (SI) was calculated by subtracting the effect of the placebo from that of benidipine at each interval. The mean SBP and DBP fell to 135 and 88 mmHg, respectively, after dosing, which gave T/P ratios of 82% and 64%, respectively. The SIs for SBP and DBP were 1.82 and 0.76, respectively. These findings indicate that benidipine maintained a satisfactory and durable antihypertensive effect by once-a-day dosing.

Screening for cardiac dysfunction in asymptomatic patients by measuring B-type natriuretic peptide levels.

Early diagnosis and treatment of heart failure lead to improved survival; pre-clinical detection would thus be beneficial. A non-invasive biochemical testing method would indeed be ideal to screen for the condition. In the present study, we sought to determine whether circulating levels of B-type natriuretic peptide (BNP) correlate with cardiac function in asymptomatic subjects. 294 consenting asymptomatic subjects were examined. BNP levels in elevated patients (> 18.4 pg / ml) showed significant correlation with echocardiographic parameters of the systolic and diastolic functions (EF r = -0.51, FS r = -0.50, E/A r = 0.42, p < 0.01). Moderate correlation with the CTR on chest X-ray was also seen (r = 0.23, p < 0.01). Multiple regression analysis showed numerous echocardiographic and hemodynamic parameters including those of systolic and diastolic function in addition to left ventricular wall thickness, blood pressure and serum creatinine levels to be significantly associated with raised BNP levels. Elevated BNP levels reflect cardiac function (both systolic and diastolic) in the asymptomatic population. Detection of cardiac dysfunction by the non-invasive biochemical test may prove useful in early pre-clinical diagnosis of heart failure.

Angiographically demonstrated coronary collaterals predict residual viable myocardium in patients with chronic myocardial infarction: a regional metabolic study.

Angiographical demonstration of coronary collateral circulation may suggest the presence of residual viable myocardium. The development of coronary collaterals was judged according to Rentrop's classification in 37 patients with old anteroseptal myocardial infarction and 13 control patients with chest pain syndrome. The subjects with myocardial infarction were divided into 2 groups: 17 patients with the main branch of the left coronary artery clearly identified by collateral blood flow from the contralateral coronary artery [Coll(+)group, male/female 10/7, mean age 56.6 years]and 20 patients with obscure coronary trunk [Coll(-)group, male/female 16/4, mean age 54.9 years]. Thallium-201 myocardial scintigraphy and examination of local myocardial metabolism were carried out by measuring the flux of lactic acid under dipyridamole infusion load. Coronary stenosis of 99% or total occlusion was found in only 5 of 20 patients (25%)in the Coll(-)group but in 16 of 17 patients(94%)in the Coll(+)group(p < 0.001). Redistribution of myocardial scintigraphy was found in 11 of 15 patients(73%)in the Coll(+)group, but only 3 of 18 patients (17%)in the Coll(-)group(p < 0.01). The myocardial lactic acid extraction rate was--13.2 +/- 17.0% in the Coll(+)group, but 9.1 +/- 13.2% in the Coll(-)group(p < 0.001). These results suggest that coronary collateral may contribute to minimizing the infarct area and to prediction of the presence of viable myocardium.

[Chemical control of cell differentiation of human myeloleukemia K562 cell line].

Human myeloid leukemia K562 cells can be induced to differentiate to mature cells bidirectionary, i.e., hemin induces erythroid differentiation, while 12-O-tetradecanoylphorbol 13-acetate (TPA) induces differentiation to monocytes. The differentiation-inducing activity of various hemin-related compounds suggested certain structural requirements for the activity: 1) the iron moiety of hemin is not essential, and 2) the propionic acid side chains of hemin play an important role in the differentiation and induction. In addition, we have examined the influence of some bioresponse-modifying factors on hemin/protoporphyrin IX-induced differentiation of K562 cell line. Retinoids and tubulin-disruptors, themselves did not induce differentiation, enhanced hemin/protoporphyrin IX-induced differentiation of K562 cells. We also examined the possible involvement of peripheral-type benzodiazepine receptor (PBR) in hemin/protoporphyrin IX-induced differentiation on K562 cell lines. The PBR specific ligands modified hemin-induced differentiation. These results suggest a requirement for retinoids (or retinoids-like cofactors) for hemin/protoporphyrin IX-induced differentiation of K562 cells and the involvement of PBR in erythroid differentiation of K562 cell line. Further we showed that TPA suppresses hemin-induced erythroid differentiation of K562 cells, while retinoids augment it. TPA is a potent inducer of heme oxygenase (HO), which catabolizes heme to biliverdin. An HO inhibitor, tin protoporphyrin (SnPP), suppresses TPA-induced K562 cell differentiation to monocytes. It was also found that cotreatment of K562 cells with SnPP and TPA induces erythroid differentiation of K562 cells, though SnPP alone or TPA alone does not induce erythroid differentiation, suggesting a role of HO in the directional switch of differentiation.

Heme deficiency in erythroid lineage causes differentiation arrest and cytoplasmic iron overload.

Erythroid 5-aminolevulinate synthase (ALAS-E) catalyzes the first step of heme biosynthesis in erythroid cells. Mutation of human ALAS-E causes the disorder X-linked sideroblastic anemia. To examine the roles of heme during hematopoiesis, we disrupted the mouse ALAS-E gene. ALAS-E-null embryos showed no hemoglobinized cells and died by embryonic day 11.5, indicating that ALAS-E is the principal isozyme contributing to erythroid heme biosynthesis. In the ALAS-E-null mutant embryos, erythroid differentiation was arrested, and an abnormal hematopoietic cell fraction emerged that accumulated a large amount of iron diffusely in the cytoplasm. In contrast, we found typical ring sideroblasts that accumulated iron mostly in mitochondria in adult mice chimeric for ALAS-E-null mutant cells, indicating that the mode of iron accumulation caused by the lack of ALAS-E is different in primitive and definitive erythroid cells. These results demonstrate that ALAS-E, and hence heme supply, is necessary for differentiation and iron metabolism of erythroid cells.

Localization of opioid-binding cell adhesion molecule (OBCAM) in adult rat brain.

We investigated the tissue distribution and brain localization of opioid-binding cell adhesion molecule (OBCAM) in the adult rats by immunoblotting and immunohistochemistry using a monoclonal anti-OBCAM peptide antibody that is specific for OBCAM. OBCAM was preferentially expressed in the central nervous system (CNS) and at a very low level in the spleen. Within the brain, OBCAM was distributed in almost all the gray matter, but little or no immunoreactive OBCAM was found in the white matter. Morphologically, the distribution pattern of OBCAM immunoreactivity was very similar to that of synaptophysin, suggesting a role in the synaptic machinery.

Characterization of an antagonist monoclonal antibody, GHBP116, specific for human growth hormone receptors.

To obtain an antagonist antibody against human growth hormone receptors (hGHRs), we prepared monoclonal antibodies against the recombinant hGHR extracellular domain. One of the clones, GHBP116, exhibited binding activity to intact human IM-9 cells and effectively immunoprecipitated the receptors in cell lysate. GHBP116 competitively inhibited 125I-human growth hormone (hGH) binding to the cells. The antagonist activity of GHBP116 was assessed in terms of ligand-induced receptor internalization, degradation, and phosphorylation of signal transducer and activator of transcription (STAT) 5. The antibody alone did not cause internalization or degradation of hGHRs, but a 1:25000 dilution of ascitic fluid almost completely inhibited ligand (1 nM hGH)-induced internalization and degradation of surface hGHRs. Moreover, GHBP116 alone did not stimulate the phosphorylation of STAT5, used as an indicator of Janus kinase (JAK)-STAT signaling, but almost completely inhibited hGH-induced phosphorylation of STAT5. These results suggest that GHBP116 acts as a specific antagonist of hGH.

Regulation of lens fiber cell differentiation by transcription factor c-Maf.

To elucidate the regulatory mechanisms underlying lens development, we searched for members of the large Maf family, which are expressed in the mouse lens, and found three, c-Maf, MafB, and Nrl. Of these, the earliest factor expressed in the lens was c-Maf. The expression of c-Maf was most prominent in lens fiber cells and persisted throughout lens development. To examine the functional contribution of c-Maf to lens development, we isolated genomic clones encompassing the murine c-maf gene and carried out its targeted disruption. Insertion of the beta-galactosidase (lacZ) gene into the c-maf locus allowed visualization of c-Maf accumulation in heterozygous mutant mice by staining for LacZ activity. Homozygous mutant embryos and newborns lacked normal lenses. Histological examination of these mice revealed defective differentiation of lens fiber cells. The expression of crystallin genes was severely impaired in the c-maf-null mutant mouse lens. These results demonstrate that c-Maf is an indispensable regulator of lens differentiation during murine development.

Modification by heme oxygenase inhibitor, tin protoporphyrin, of cellular differentiation of human myeloid leukemia K562 cell line.

Human myeloid leukemia K562 cells can be induced to differentiate to mature cells bidirectionally, i.e., hemin induces erythroid differentiation, while 12-O-tetradecanoylphorbol 13-acetate (TPA) induces differentiation to monocytes. TPA is also a potent inducer of heme oxygenase (HO), which catabolizes heme to biliverdin. We show here that TPA suppresses hemin-induced erythroid differentiation of K562 cells, while retinoids augment it. Further, an HO inhibitor, tin protoporphyrin (SnPP), suppresses TPA-induced K562 cell differentiation to monocytes. It was also found that co-treatment of K562 cells with SnPP and TPA induces erythroid differentiation of K562 cells, though SnPP alone or TPA alone does not induce erythroid differentiation, suggesting a role of HO in the directional switch of differentiation.

Role of GATA-1 in proliferation and differentiation of definitive erythroid and megakaryocytic cells in vivo.

To elucidate the contributions of GATA-1 to definitive hematopoiesis in vivo, we have examined adult mice that were rendered genetically defective in GATA-1 synthesis (Takahashi et al, J Biol Chem 272:12611, 1997). Because the GATA-1 gene is located on the X chromosome, which is randomly inactivated in every cell, heterozygous females can bear either an active wild-type or mutant (referred to as GATA-1.05) GATA-1 allele, consequently leading to variable anemic severity. These heterozygous mutant mice usually developed normally, but they began to die after 5 months. These affected animals displayed marked splenomegaly, anemia, and thrombocytopenia. Proerythroblasts and megakaryocytes massively accumulated in the spleens of the heterozygotes, and we showed that the neomycin resistance gene (which is the positive selection marker in ES cells) was expressed profusely in the abnormally abundant cells generated in the GATA-1.05 mutant females. We also observed hematopoiesis outside of the bone marrow in the affected mutant mice. These data suggest that a small number of GATA-1.05 mutant hematopoietic progenitor cells begin to proliferate vigorously during early adulthood, but because the cells are unable to terminally differentiate, this leads to progenitor proliferation in the spleen and consequently death. Thus, GATA-1 plays important in vivo roles for directing definitive hematopoietic progenitors to differentiate along both the erythroid and megakaryocytic pathways. The GATA-1 heterozygous mutant mouse shows a phenotype that is analogous to human myelodysplastic syndrome and thus may serve as a useful model for this disorder.

Stimulation of hydrogen peroxide production by drinking water contaminants in HL-60 cells sensitized by retinoic acid.

Chemical carcinogens, such as chloroform and trichloroethylene, are present in drinking water in Japan. As these contaminants are believed to have a role in carcinogenesis, we examined if chloroform and trichloroethylene, as well as methylene chloride, xylene, benzene, and ethanol, have the ability to generate hydrogen peroxide (H(2)O(2)) in human polymorphonuclear leukocytes (PMN) and human leukemia (HL-60) cells. Methylene chloride, benzene, xylene, trichloroethylene, and ethanol did not increase cellular H(2)O(2): production as measured by flow cytometry nor as observed by confocal laser microscopy. In PMN and RAuntreated HL-60 cells chloroform did not significantly affect H(2)O(2) levels. However, in HL-60 cells sensitized by pretreatment of 10 nM retinoic acid (RA) for 12 h, chloroform induced a significant increase in H(2)O(2), but the increase induced by trichloroethylene was not significant. The observed increase in fluorescence was confirmed using a confocal laser microscope. These results indicate that chloroform and trichloroethylene may stimulate H(2)O(2) production in HL60 cells sensitized by pretreatment of RA. Our method may be useful to test if weak stimulants can stimulate intracellular H(2)O(2) production.

Expression of opioid-binding cell adhesion molecule (OBCAM) and neurotrimin (NTM) in E. coli and their reactivity with monoclonal anti-OBCAM antibody.

Opioid-binding cell adhesion molecule (OBCAM), neurotrimin (NTM) and limbic system-associated membrane protein (LAMP) are homologous and are the members of the IgLON family which is a subfamily within the immunoglobulin superfamily. We cloned the cDNAs for OBCAM and NTM, prepared recombinant proteins, and examined the reactivity of the previously prepared monocolonal anti-OBCAM antibody, OBC53, with the recombinant proteins by immunoblotting. These experiments revealed that OBC53 recognizes OBCAM about 1000 times as efficiently as NTM. Moreover, the NTM and LAMP peptides which have sequences homologous to the OBCAM peptide used for the preparation of OBC53 were 150 times less reactive to OBC53. Thus, the OBC53 antibody is a useful tool for specifically detecting OBCAM in immunochemical experiments.