A neuron-specific isoform of brain ankyrin, 440-kD ankyrinB, is targeted to the axons of rat cerebellar neurons.

Two isoforms of brain ankyrin, 440- and 220- kD ankyrinB, are generated from the same gene by alternative splicing of pre-mRNA. The larger isoform shares the same NH2-terminal and COOH-terminal domains to the smaller isoform and contains, in addition, a unique inserted domain of about 220-kD in size (Kunimoto, M., E. Otto, and V. Bennett. 1991. J. Cell Biol. 115:1319-1331). Both Isoforms were expressed in primary cerebellar cells in a manner similar to that in vivo; the larger isoform appeared first when axogenesis is actively conducted and the smaller isoform came up later. 440-kD ankyrinB was localized in the axons of cerebellar neurons both in vivo and in vitro using an antibody raised against the insert region, while 220-kD isoform was rather localized in the cell bodies and dendrites of neurons by a specific antibody prepared using a synthetic peptide corresponding to the splice site as antigen. Astroglia cells also expressed 220-kD ankyrinB but not the 440-kD isoform. These results indicate that 440-kD ankyrinB is a neuron-specific isoform targeted to the axons and its unique inserted domain is essential for the targeting.

A new 440-kD isoform is the major ankyrin in neonatal rat brain.

This report describes initial characterization of a 440-kD isoform of brain ankyrin (ankyrinB) representing an alternatively spliced mRNA product of the gene encoding the major isoform of ankyrin in adult human brain (Otto, E., M. Kunimoto, T. McLaughlin, V. Bennett, J. Cell Biology. 114:241-253). Northern and immunoblot analyses indicate that 440-kD ankyrinB includes the spectrin and membrane-binding domains as well as a regulatory domain of the major 220-kD isoform. 440-kD ankyrinB contains, in addition, a sequence of a predicted size of 220 kD which is inserted between the regulatory domain and spectrin/membrane-binding domains. 440-kD ankyrinB has properties expected of a peripherally associated membrane-skeletal protein: it is exclusively present in the particulate fraction of brain homogenates, is extracted with NaOH, and remains associated with Triton-X-100-resistant structures. Expression of 440-kD ankyrinB in rat brain began at birth before other ankyrins could be detected, peaked 10 d after birth, and then decreased progressively to 30% of the maximum in adults. Expression of the 220-kD ankyrinB and ankyrinR (erythroid ankyrin) began approximately 10 d after the 440-kD isoform, increased rapidly between 10 and 15 d after birth, and finally achieved their maximal levels in adults. 440-kD ankyrinB is present in approximately equivalent amounts in all regions of neonatal brain while in adult brain it is present in highest levels in cerebellum and lowest in brain stem. 440-kD ankyrinB was localized by immunofluorescence in regions of neonatal and adult brain containing primarily dendrites and unmyelinated axons. 440-kD ankyrinB thus may play a specialized role in neuronal processes.

Isolation and characterization of cDNAs encoding human brain ankyrins reveal a family of alternatively spliced genes.

Ankyrins are a family of membrane-associated proteins that can be divided into two immunologically distinct groups: (a) erythrocyte-related isoforms (ankyrinR) that have polarized distributions in particular cell types; and (b) brain-related isoforms (ankyrinB) that display a broader distribution. In this paper, we report the isolation and sequences of cDNAs related to two ankyrinB isoforms, human brain ankyrin 1 and 2, and show that these isoforms are produced from alternatively spliced mRNAs of a single gene. Human brain ankyrin 1 and 2 share a common NH2-terminus that is similar to human erythrocyte ankyrins, with the most striking conservation occurring between areas composed of a repeated 33-amino acid motif and between areas corresponding to the central portion of the spectrin-binding domain. In contrast, COOH-terminal sequences of brain ankyrin 1 and 2 are distinct from one another and from human erythrocyte ankyrins, and thus are candidates to mediate protein interactions that distinguish these isoforms. The brain ankyrin 2 cDNA sequence includes a stop codon and encodes a polypeptide with a predicted molecular mass of 202 kD, which is similar to the Mr of the major form of ankyrin in adult bovine brain membranes. Moreover, an antibody raised against the conserved NH2-terminal domain of brain ankyrin cross-reacts with a single Mr = 220 kD polypeptide in adult human brain. These results strongly suggest that the amino acid sequence of brain ankyrin 2 determined in this report represents the complete coding sequence of the major form of ankyrin in adult human brain. In contrast, the brain ankyrin 1 cDNAs encode only part of a larger isoform. An immunoreactive polypeptide of Mr = 440 kD, which is evident in brain tissue of young rats, is a candidate to be encoded by brain ankyrin 1 mRNA. The COOH-terminal portion of brain ankyrin 1 includes 15 contiguous copies of a novel 12-amino acid repeat. Analysis of DNA from a panel of human/rodent cell hybrids linked this human brain ankyrin gene to chromosome 4. This result, coupled with previous reports assigning the human erythrocyte ankyrin gene to chromosome 8, demonstrates that human brain and erythrocyte ankyrins are encoded by distinct members of a multigene family.

Toxicity assessment of the extract of compost as a final product from Bio-Toilet.

Bio-Toilet is the name of a dry closet or composting toilet using sawdust as an artificial soil matrix for bioconversion of human excrement into compost. Since feces and urine contain several chemicals such as pharmaceutical residues and endocrine disruptors and they may still remain in compost after biological reaction in the Bio-Toilet, it is required to examine the possibility of soil and/or groundwater pollution by applying compost to a soil system in farmland. In this study, toxicity of Bio-Toilet compost was evaluated by measuring the viability of human neuroblast (NB-1). The bio-assay was applied to the water extract of compost from the Bio-Toilets which are in practical use in Japan. The assay results showed that (1) the extract of feces showed no toxicity, and the extracts of unused sawdust had no or low level toxicity and (2) the extracts of composts had heavier toxicity than unused sawdust. These results implied that some chemicals that have toxicity were generated by biological reactions or accumulated in toilet system. The bioassay results with fractionated organic matter by its molecular weight showed that the small molecular weight fraction had stronger toxicity than other fractions. The effect of inorganic matter on toxicity was examined by comparing the dose-response relationship of the extracts of compost with positive control with 1M of sodium chloride solution. The comparison showed that sodium concentration in the extract was too low to develop the toxicity and the effect of inorganic matter could be neglected in this study.

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium.

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.

p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats.

Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.

Role of hydrophilic organic matter on developing toxicity in decay process of activated sludge.

It is known that the toxicity of effluent is more intensive than that of influent in the activated sludge process. In this study, we applied bioassay using cultured human cell lines to the decay process of activated sludge to evaluate the toxicity of organic matter generated and/or released from activated sludge bacteria. We also applied this bioassay to hydrophilic fraction of samples. The bioassay results showed that: (1) the variation in the dose-response relation obtained from assay with original samples was observed during decay; (2) on the other hand, the response curves of only hydrophilic fraction at each time show the same relationship between TOC and viability of MCF7 cells; (3) this trend was confirmed by plotting the time course of EC50. These results imply that: (1) the hydrophilic organic matter controlled for developing toxicity during decay process of activated sludge; and (2) the character of hydrophilic organic matter is not changed during the experimental period.

Possible genetic influence on the strength of human muscle nerve sympathetic activity at rest.

Large reproducible interindividual differences in the strength of human muscle nerve sympathetic activity have been demonstrated previously without satisfactory explanation. We undertook the present study to investigate whether a genetic influence may be a factor of importance. Microneurographic recordings of sympathetic impulse traffic were made in the peroneal nerve in nine pairs of monozygotic male twins and eight pairs of age-matched male subjects without family relationship. The strength of the sympathetic activity was quantitated as number of sympathetic bursts per 100 heart beats and bursts per minute. Group mean values of muscle sympathetic activity, heart rate, and blood pressure were similar in the two groups. Intrapair differences (mean +/- SEM) of sympathetic activity were 5.4 +/- 1.7 bursts per 100 heart beats (1.7 +/- 0.5 bursts per minute) for the twins and 19.4 +/- 3.2 bursts per 100 heart beats (11.8 +/- 2.5 bursts per minute) for the control subjects (P < .01 for both). The degree of reproducibility between twins is similar to that reported previously between repeated recordings in the same subject. The finding may indicate that the strength of sympathetic outflow to muscle is controlled genetically. If so, we speculate that this may contribute to the heritability of blood pressure in both normotensive and hypertensive subjects.

Effects of unilateral lesion of the nucleus basalis of Meynert on brain glucose utilization in callosotomized baboons: a PET study.

Prior work has demonstrated that unilateral lesions of the nucleus basalis of Meynert (NbM) in baboons induce a marked reduction in glucose utilization of the ipsilateral cerebral cortex, linearly proportional to the depression in cortical choline acetyltransferase (ChAT) activity achieved. Unexpectedly, there was also marked hypometabolism of the contralateral cerebral cortex, and glucose utilization recovered gradually on both sides despite persistent deficit in cortical ChAT activity. To investigate the role of the corpus callosum (CC) in this bilateral metabolic effect and subsequent recovery, three baboons were subjected to unilateral electrolytic NbM lesion greater than 3 months following section of the anterior CC. Brain glucose utilization was sequentially studied by positron emission tomography; ChAT activity was measured and histological sections obtained after death. In these animals, the NbM lesion also induced significant metabolic depression over the ipsilateral cortex, proportional to the reduction in ChAT activity. Corpus callosotomy did not prevent the contralateral metabolic effects, suggesting that the latter do not normally operate through the CC. However, there was no significant recovery of glucose utilization, suggesting that, following unilateral NbM lesion, the CC normally mediates, at least in part, the recovery of cortical glucose utilization.

Possible involvement of the 440 kDa isoform of ankyrinB in neuritogenesis in human neuroblastoma NB-1 cells.

Two isoforms of brain ankyrin, 440 kDa and 220 kDa ankyrinB, which are products of alternatively spliced pre-mRNA encoded by a single gene, are both expressed in human neuroblastoma NB-1 cells. Expression of the polypeptide and mRNA of the larger isoform increased upon induction of neurite outgrowth in NB-1 cells by dibutyryl cAMP, while those of the smaller isoform were unaffected. The expressed 440 kDa ankyrinB was concentrated at the tip of growing neurites and was partly co-localized with GAP-43. These results suggest that 440 kDa ankyrinB is one of the neuronal growth-associated proteins and provides an interesting example of gene regulation by alternative splicing in neuronal cells.

Effect of L-threo-3,4-dihydroxyphenylserine on muscle sympathetic nerve activities in Shy-Drager syndrome.

We observed changes in postganglionic efferent discharges of muscle sympathetic nerve (muscle sympathetic activity, MSA) microneurographically before and after the oral administration of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS), a precursor of norepinephrine, in a patient with Shy-Drager syndrome and irregular fluctuations of blood pressure. Before drug administration, MSA was only rarely observed with the patient in the supine position. There was a slight increase in MSA during head-up tilting to 40 degrees, and orthostatic hypotension (OH) occurred just after the body was tilted head upward to 40 degrees. MSA became prominent 30 minutes after the oral administration of 200 mg of L-threo-DOPS while the patient was in a 40 degree head-up position, and the OH was improved. The MSA discharge rate decreased and OH reappeared 3 hours after oral administration, when the plasma concentration of norepinephrine was at its highest level. We suggest that the OH improved mainly because of the increase in MSA due to L-threo-DOPS, and that the drug may activate sympathetic outflow at a site proximal to the sympathetic ganglion.

Anticonvulsant activity of the diaryltriazine, LY81067: studies using electroencephalographic recording and positron emission tomography.

It is reported that LY81067, a new diaryltriazine, possesses anticonvulsant properties against grand mal status epilepticus induced by intravenous administration of picrotoxin binding site ligands (Ro 5-4864 and pentylenetetrazole) in the baboon. Intravenous administration of LY81067 during the seizures blocked grand mal type electroencephalographic (EEG) paroxysmal discharges and led to a long electrical silence, progressively replaced by spike-and-wave discharges of low frequency (2 c/sec). A transient blocking effect was also observed when LY81067 was injected during grand mal status epilepticus induced by the benzodiazepine inverse agonist methyl beta-carboline-3-carboxylate; however, the long electrical silence observed after administration of LY81067 was rapidly followed by grand mal type paroxysmal discharges in the EEG, which could be stopped by a subsequent injection of Ro 15-1788. However, LY81067 also displayed intrinsic epileptogenic properties. Administration of this drug alone led to the appearance of rhythmic EEG (2-3 c/sec) associated with myoclonia. Concomitantly with the EEG studies, interactions of all these drugs with benzodiazepine receptors were observed in vivo using [11C]Ro 15-1788 as radioligand and positron emission tomography (PET) as a non-invasive technique to measure the binding of the [11C]benzodiazepine antagonist in brain, in vivo. The [11C]Ro 15-1788 bound in the brain could not be displaced by the administration of LY81067 but rather, the [11C]antagonist binding in the brain was somewhat enhanced. Administration of pentylenetetrazole or Ro 5-4864 decreased the rate of wash-out of the radioligand. This fast effect of these two convulsant drugs was partially inhibited by the subsequent administration of LY81067. The concomitant blocking of the grand mal status epilepticus was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of 5' upstream promoter region of prostacyclin synthase gene variant with cerebral infarction.

The aim of this study was to investigate whether there is an association between the promoter region of the prostacyclin synthase gene and cerebral infarction (CI). Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we found a variable-number tandem repeat polymorphism in the 5'-upstream promoter region of the prostacyclin synthase gene. This region contains transcriptional factors-binding sites of Spl (CCCGCC) and AP-2 (CCGCCAGCCCC). The alleles varied in size from three to seven repeats of nine base pairs (bp). We performed an association study using the polymorphism in 111 patients and 152 control subjects. The transcriptional activity of the abnormal promoter region allele was determined by luciferase assay. The overall distribution of alleles differed significantly between both groups. Logistic linear regression analysis revealed the small number repeat allele to be found more frequently with CI. Transcriptional activity increased with increasing numbers of repeats. This study provides consistent support for the association between CI and the PGIS gene.

Polymorphism of the promoter region of prostacyclin synthase gene is not related to essential hypertension.

An impaired synthesis of prostacyclin has been implicated in the development of essential hypertension (EH). We therefore investigated whether there is an association between the prostacyclin synthase (PGIS) gene and EH using a variable number of tandem repeats (VNTR) polymorphism in the promoter region that influences transcriptional activity of this gene. A total of 125 patients with EH and 125 age-matched subjects with normal blood pressure were studied. The number of VNTR of the five alleles ranged from 3 to 7 repeats in the 250 unrelated Japanese subjects. The allele frequency distribution in the two groups were not significantly different. Thus, this VNTR polymorphism in the PGIS gene is not associated with EH.

Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction.

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.

Comparison of the cytoskeleton fractions of rat red blood cells prepared with non-ionic detergents.

The characteristics of cytoskeleton fractions prepared from rat red cell ghosts with four non-ionic detergents were studied. One percent (w/v) solutions of Triton X-100, Emulgen 911, MEGA-9 (nonanoyl-N-methylglucamide), and octylglucoside solubilized 78, 68, 80, and 92% of the ghost phospholipid, while they solubilized 82, 78, 72, and 62% of the ghost band 3, a transmembrane protein, respectively. There was no correlation between the solubilization percentages of phospholipid and band 3. Phospholipids retained in cytoskeleton fractions were shown to exist as blebs on the surface by electron microscopic observation. The cytoskeleton fraction prepared with octylglucoside retained about two-fold more band 3 than that with Triton X-100 (Triton shells). However, cytoskeleton fractions prepared from p-chloromercuribenzoate-treated ghosts with the two detergents retained almost equal amounts of band 3, less than 5% of that in the ghosts. Under this condition, most of band 2.1, a protein linking band 3 to the spectrin-actin network, was released from the cytoskeleton fractions. The band 3 solubilized with octylglucoside sedimented faster in a linear sucrose gradient and had a larger Stokes' radius than that with Triton X-100, which is known to exist as dimer. These results strongly suggest that octylglucoside does not disturb the association of tetrameric band 3 with the spectrin-actin network, while Triton X-100 dissociates tetrameric band 3 to the dimer, resulting in the difference in the amount of band 3 retained in cytoskeleton fractions. In conclusion, octylglucoside can produce a more native cytoskeleton fraction of red cell membranes than Triton shells.

Antibody against ganglioside GD1c containing NeuGcalpha2-8NeuGc cooperates with CD3 and CD4 in rat T cell activation.

Gangliosides have long been implicated in T cell activation. GD1c with two N-glycolylneuraminic acids [GD1c(NeuGc,NeuGc)] is the predominant ganglioside in rat T cells. In the present study, the anti-GD1c(NeuGc,NeuGc) mAb, AC1, which binds to the NeuGcalpha2-8NeuGcalpha2- sequence, was found to enhance Con A-activated cellular proliferation at a concentration at which AC1 alone did not activate the cells. The potentiation by AC1 was observed more consistently and effectively in the cellular activation elicited by cross-linking of anti-CD3 and anti-CD4, rather than in the cell growth induced by immobilized anti-CD3 alone. Moreover, the combination of immobilized anti-CD4 and soluble AC1 had a remarkable mitogenic effect. In addition, we have demonstrated the existence of a 100 kDa protein in rat T cell lysates which reacts with AC1 on Western blots, and this interaction is abolished by sialidase-treatment of the membrane. Pronase treatment of the T cells, which rendered the 100 kDa protein undetectable on Western-blotting, reduced the number of AC1-positive cells by 40-50% on flow cytometry. On the other hand, all cells became AC1-negative after sialidase treatment. These findings indicated that AC1 reacts with both GD1c(NeuGc,NeuGc) and the 100 kDa glycoprotein on rat T cells. Taken together, these results predict the presence of a novel regulatory mechanism of T cell activation involving CD4 and the NeuGcalpha2-8NeuGcalpha2- sequence.

Nitric oxide inactivates glyoxalase I in cooperation with glutathione.

We previously found that glyoxalase I (Glo I) is inactivated upon exposure of human endothelial cells to extracellular nitric oxide (NO), and this event correlates with an increase in its pI on two-dimensional gels. In this study, we demonstrate that NO can modulate Glo I activity in cooperation with cellular glutathione (GSH). Severe depletion of intracellular GSH prevents the inactivation of Glo I in response to NO, although such depletion enhances the inactivation of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a well-known enzyme susceptible to NO-induced oxidation. S-Nitrosoglutathione (GSNO), an adduct of GSH and NO, lowers the activity of purified human Glo I, while S-nitrosocysteine (CysNO) inactivates the enzyme only in the presence of GSH. This indicates that a dysfunction in Glo I would require the formation of GSNO in situ. Competitive inhibitors of Glo I, S-(4-bromobenzyl)glutathione and its membrane-permeating form, completely abolish the NO action in vitro and inside cells, respectively. Taken together, these results reveal that Glo I can interact directly with GSNO, and that the interaction converts Glo I into an inactive form. Moreover, the data suggest that the substrate recognition site of Glo I might be involved in the interaction with GSNO.

Selective down-regulation of 440 kDa ankyrinB associated with neurite retraction.

Brain-specific isoforms of ankyrin, 440 kDa and 220 kDa ankyrinB, which are generated from a single gene by alternative splicing of pre-mRNA, are both expressed in human neuroblastoma NB-1 cells and the expression of the larger isoform is increased upon induction of neurite outgrowth. Exposure to methylmercury, a potent neurotoxic substance, at a sublethal dose induced dramatic retraction of neurites in NB-1 cells. Concomitantly, synthesis of 440 kDa ankyrinB polypeptide and mRNA were selectively attenuated in methylmercury-treated cells, while the 220 kDa isoform was not affected. These results indicate that the expression of 440 kDa ankyrinB is intimately associated not only with the neurite outgrowth but also with neurite retraction in neuronal cells, and is regulated at mRNA level.

Association study between the variants of the human ANP gene and essential hypertension.

Variants of atrial natriuretic peptide (ANP) are reported to be more common in blacks with hypertension than in normotensive controls and constitute an independent risk factor for cerebral infarction. The purpose of the present study was to investigate the role of ANP in the pathogenesis of essential hypertension (EH) in the Japanese. We investigated 2 previously reported ANP gene markers, G1837A and T2238C, for their possible associations with EH. A total of 233 individuals with EH and 213 age-matched normotensive (NT) control subjects were studied. The frequencies of the G and A alleles were 0.09 (42/466) and 0.91 (424/466), respectively, for the NT group and 0.11 (47/426) and 0.89 (379/426), respectively, for the EH group. These frequencies did not differ significantly between the two groups. The frequencies of the T and C alleles were 0.024 (11/466) and 0.97 (455/466), respectively, for the NT group and 0.03 (13/426) and 0.97 (413/426), respectively, for the EH group. These frequencies also did not differ significantly between the two groups. Neither G1837A nor the T2238C polymorphism of the ANP gene was associated with EH. Our findings do not support the hypothesis that the G1837A and T2238C polymorphisms of the ANP gene are markers for EH in the Japanese.