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Biomedical subjects

K Kikuchi

Publications and source records attributed to K Kikuchi.

At least 289 records · Page 16Linked to original sources
PubMed1999

Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide.

The effects of airway (AH) and vascular hypoxia (VH) on the production of nitric oxide (NO; VNO) were tested in isolated buffer-perfused (BFL) and blood-perfused rabbit lungs (BLL). To produce AH and/or VH, the lung was ventilated with 1% O(2) gas, and/or the perfusate was deoxygenated by a membrane oxygenator located on the inlet limb to the pulmonary artery. We measured exhaled NO (VNO), accumulation of perfusate NOx, and pulmonary arterial pressure (Ppa) during AH (inspired O(2) fraction = 0.01) and/or VH (venous PO(2) = 26 Torr). In BFL, a pure AH without VH caused decreases in VNO and NOx accumulation with a rise in Ppa. However, neither VNO, NOx accumulation, nor Ppa changed during VH. Similarly, in BLL, only AH reduced VNO, although NOx accumulation was not measurable because of Hb. When alveolar PO(2) was gradually reduced from 152 to 0 Torr for 20 min, AH reduced VNO curvilinearly from 73.9 +/- 8 to 25.6 +/- 8 nl/min in BFL and from 26.0 +/- 2 to 5. 2 +/- 1 nl/min in BLL. This plot was analogous to that of a substrate-velocity curve for an enzyme obeying Michaelis-Menten kinetics. The apparent Michaelis-Menten constant for O(2) was calculated to be 23.2 microM for BLL and 24.1 microM for BFL. These results indicate that the VNO in the airway epithelia is dependent on the level of inspired O(2) fraction, leading to the tentative conclusion that epithelial NO synthase is O(2) sensitive over the physiological range of alveolar PO(2) and controls pulmonary circulation.

Animals↗
PubMed1999

Sonic motor nucleus and its connections with octaval and lateral line nuclei of the medulla in a rockfish, Sebastiscus marmoratus.

The sonic motor nucleus and its fiber connections were examined in a rockfish, Sebastiscus marmoratus by means of tracer methods using horseradish peroxidase (HRP), biocytin, and carbocyanine dye (DiI). Sebastiscus has a swimbladder and a pair of extrinsic sonic/drumming muscles. The sonic muscle is ipsilaterally innervated by the occipital nerve which is composed of two ventral roots arising from the sonic motor nucleus. The sonic motor neurons are distributed in the most ventral part of the ventral column from the caudal medulla to the rostral spinal cord, and form a ventrally located columnar nucleus. Each neuron in this nucleus possesses a long thick dendrite and several short dendrites. The long dendrite extends dorsolaterally and branches in the lateral funiculus, whereas the short dendrites branch around their cell bodies. After biocytin injections into the sonic motor nucleus, two groups of premotor neurons were retrogradely labeled bilaterally, one in the dorsomedial portion of the descending octaval nucleus (DO) and the other in the medial zone of the reticular formation (RF) in the medulla. The DO premotor neurons were multipolar with several dendrites branching near the cell bodies, and the RF premotor neurons were bipolar. One of the two dendrites of the RF premotor neurons extends laterally into the ventral portion of the DO, and the other dendrite extends into the ventromedial area in the medulla. In the ventromedial dendritic field of the RF premotor neurons, descending fibers arising from the optic tectum (TO) and torus semicircularis (TS) traverse in the tractus tectobulbaris and terminate bilaterally. After DiI insertion into the ventromedial dendritic field, retrogradely labeled neurons were found bilaterally in the TS and TO. The majority of tectal neurons were located in the stratum griseum centrale. These neurons had two short basal dendrites branching in the cell layer and a long apical dendrite extending to the stratum fibrosum et griseum superficiale and stratum opticum. The toral neurons were bipolar and were distributed throughout the TS. Furthermore, biocytin injections into the medial nucleus of the lateral line system revealed that the nucleus projects bilaterally to the RF premotor neurons. These results show that premotor neurons for the sonic motor nucleus are located in the dorsomedial portion of the DO and the medial zone of the RF in the medulla. It is suggested that the sonic motor nucleus receives auditory input via the DO premotor neurons and input from RF premotor neurons which receive lateral line input via the medial nucleus, vestibular input through the lateral dendrite extending into the ventral portion of the DO, and information from the TO and TS via the tractus tectobulbaris.

Air Sacs↗
PubMed1999

Genomic organization and chromosome location of the rat CD3zeta locus (Cd3z).

The structure and pattern of expression of the CD3zeta chain have apparently been conserved throughout mammalian evolution. The organization of the rat CD3zeta locus was determined by genomic cloning and nucleotide sequencing. Most of the rat CD3zeta coding region was similar to mouse and human CD3zeta sequences. Whereas the 3' region involving alternative splicing was relatively well conserved at the nucleotide level, the deduced amino acid sequences were different in rats, mice and humans due to the presence of deletion and insertion mutations. Alternative splicing products of the CD3zeta locus, such as CD3eta, CD3θ and CD3tau, which have been reported for mice, were not expressed by rat T cells. By using fluorescence in situ hybridization, we have localized rat CD3zeta to chromosome 13q22-->q23.

Alternative Splicing↗
PubMed1999

A splicing mutation of the tyrosinase gene causes yellow oculocutaneous albinism in a Japanese patient with a pigmented phenotype.

BACKGROUND: Yellow oculocutaneous albinism (OCA) that is caused by tyrosinase gene mutations shows two characteristics: extreme hypopigmentation at birth and the eventual development of yellow or blond hair. OBJECTIVE: We studied a Japanese girl who had brown hair, a lighter skin color than her unaffected family and brown eyes at 9 months of age. METHODS: We performed direct sequencing analyses of the tyrosinase gene in her genomic DNA. RESULTS: The patient was a compound heterozygote for the +DeltaC310 mutation (known to result in absent melanogenic activity) and a second t-->a transition at the 3' end of intron 2. CONCLUSION: The t-->a transition has previously been reported as a splicing mutation in other Caucasian patients with a typical yellow OCA phenotype. However, this patient showed much more pigmentation than that reported in Caucasians. Therefore, we estimate that the mild phenotype results from her genetic pigment background.

Albinism, Oculocutaneous↗
PubMed1999

Effects of vasodilatory beta-adrenoceptor antagonists on endothelium-derived nitric oxide release in rat kidney.

The mechanisms for the vascular actions of vasodilatory beta-blockers remain undetermined. For some kinds of beta-blockers, the involvement of nitric oxide (NO) has been suggested. We studied the effects of vasodilatory beta-blockers on renal perfusion pressure (RPP) and NO release in the rat kidney. Infusion of bopindolol, celiprolol, and nebivolol caused a dose-dependent reduction in RPP and an increase in NO release (RPP: bopindolol 10(-6) mol/L, -23+/-2%; celiprolol 10(-4) mol/L, -27+/-2%; nebivolol 10(-5) mol/L, -35+/-3%; NO: bopindolol 10(-6) mol/L, +33+/-2; celiprolol 10(-4) mol/L, +41+/-2; nebivolol 10(-5) mol/L, +45+/-5 fmol. min-1. g kidney-1, mean+/-SEM). Metergoline (10(-6) mol/L), a 5-hydroxytryptamine (5-HT)1/2 antagonist, or NAN-190 (10(-6) mol/L), a 5-HT1A antagonist, almost completely abolished the vasorelaxation and NO release caused by bopindolol, celiprolol, and nebivolol. However, neither propranolol nor bisoprolol decreased RPP. Celiprolol and nebivolol caused vasodilation in the rat thoracic aorta, and it was markedly reduced by endothelial denudation, Nomega-nitro-L-arginine methyl ester (10(-4) mol/L), or NAN-190 (10(-6) mol/L). In deoxycorticosterone acetate-salt hypertensive rats, 4-week administration of celiprolol (50 mg. kg-1. d-1 IV) restored the responses regarding RPP and NO release to acetylcholine. These results suggest that several beta-blockers exert their vasodilatory action through the 5-HT1A receptor/NO pathway and that treatment with these beta-blockers may protect against endothelial injury in hypertension.

Adrenergic beta-Antagonists↗
PubMed1999

Role of nitric oxide-cGMP pathway in adrenomedullin-induced vasodilation in the rat.

We previously reported that adrenomedullin (AM), a potent vasodilator peptide discovered in pheochromocytoma cells, stimulates nitric oxide (NO) release in the rat kidney. To further investigate whether the NO-cGMP pathway is involved in the mechanisms of AM-induced vasodilation, we examined the effects of E-4021, a cGMP-specific phosphodiesterase inhibitor, on AM-induced vasorelaxation in aortic rings and perfused kidneys isolated from Wistar rats. We also measured NO release from the kidneys using a chemiluminescence assay. AM (10(-10) to 10(-7) mol/L) relaxed the aorta precontracted with phenylephrine in a dose-dependent manner. Denudation of endothelium (E) attenuated the vasodilatory action of AM (10(-7) mol/L AM: intact (E+) -25.7+/-5.2% versus denuded (E-) -7. 8+/-0.6%, P<0.05). On the other hand, pretreatment with 10(-8) mol/L E-4021 augmented AM-induced vasorelaxation in the intact aorta (-49. 0+/-7.9%, P<0.05) but not in the denuded one. E-4021 also enhanced acetylcholine (ACh)-induced vasorelaxation in the rat intact aorta (10(-7) mol/L ACh -36.6+/-8.4% versus 10(-8) mol/L E-4021+10(-7) mol/L ACh -62.7+/-3.1%, P<0.05). In perfused kidneys, AM-induced vasorelaxation was also augmented by preincubation with E-4021 (10(-9) mol/L AM -15.4+/-0.6% versus 10(-8) mol/L E-4021+10(-9) mol/L AM -23.6+/-1.2%, P<0.01). AM significantly increased NO release from rat kidneys (DeltaNO: +11.3+/-0.8 fmol. min-1. g-1 kidney at 10(-9) mol/L AM), which was not affected by E-4021. E-4021 enhanced ACh-induced vasorelaxation (10(-9) mol/L ACh -9.7+/-1.7% versus 10(-8) mol/L E-4021+10(-9) mol/L ACh -18.8+/-2.9%, P<0.01) but did not affect ACh-induced NO release from the kidneys. In the aorta and the kidney, 10(-4) mol/L of NG-nitro-L-arginine methyl ester, an NO synthase inhibitor, and 10(-5) mol/L of methylene blue, a guanylate cyclase inhibitor, reduced the vasodilatory effect of AM. These results suggest that the NO-cGMP pathway is involved in the mechanism of AM-induced vasorelaxation, at least in the rat aorta and kidney.

Adrenomedullin↗
PubMed1999

Characteristics of intracardiac potentials at successful ablation sites in concealed Wolff-Parkinson-White syndrome.

The authors investigated bipolar electrograms recorded from the catheter tip at the actual successful ablation sites in 22 consecutive patients with concealed Wolff-Parkinson-White syndrome to clarify the characteristics of the potentials indicating the optimal site for catheter ablation. In all patients the retrograde transaortic approach to their left-sided accessory pathways, and a temperature-controlled (60 degrees C) energy delivery, were performed. The authors assumed that a shorter dissociation time (time from energy delivery to ventriculoatrial conduction dissociation) indicated more accurate catheter mapping. A significant negative correlation (r = 0.527, p < 0.05) between the AV ratio (ratio of the amplitudes of the atrial to ventricular potentials) recorded at the ablation catheter tip and the dissociation time was observed. When the AV ratio and the dissociation time were compared among the groups classified according to the corresponding Npeak (the number of positive potential peaks in the electrogram obtained from the ablation catheter tip during right ventricular apical pacing) value, they differed significantly (p < 0.05 and p < 0.01, respectively), ie, a higher AV ratio and a shorter dissociation time related to a multipeak electrogram from the ablation catheter tip. The authors conclude that the atrial insertion site of the accessory pathway, exhibiting a multipeak complex electrogram that may represent nonuniform anisotropic characteristics, is an adequate ablation site.

Action Potentials↗
PubMed1999

Depression of peripheral chemosensitivity by a dopaminergic mechanism in patients with obstructive sleep apnoea syndrome.

In the present study, respiratory drives to chemical stimuli and peripheral chemosensitivity were evaluated in patients with obstructive sleep apnoea (OSAS). The effects of oral administration of domperidone, a selective dopamine D2-receptor antagonist, were also examined, to study the respiratory effects of endogenous dopamine on peripheral chemoreceptors. Sixteen patients with OSAS and nine normal control subjects were studied. Respiratory responses to hypercapnia and hypoxia were measured using the rebreathing method and isocapnic progressive hypoxia method, respectively. The hypoxic withdrawal test, which measures the decrease in ventilation caused by two breaths of 100% O2 under mild hypercapnic hypoxic conditions (end-tidal oxygen and carbon dioxide tensions approximately 8.0 kPa and 5.3-6.7 kPa, respectively), was used to evaluate peripheral chemosensitivity. In the patients with OSAS, ventilatory responses to hypercapnia and hypoxia were significantly decreased compared with those of control subjects. Hypoxic withdrawal tests showed that peripheral chemosensitivity was significantly lower in patients with OSAS than in normal subjects. Hypercapnic ventilatory response and peripheral chemosensitivity were enhanced by administration of domperidone in the patients with OSAS, although no changes in either of these were observed in the control subjects. The hypoxic ventilatory response and peripheral chemosensitivity in the patients with OSAS were each significantly correlated with severity of hypoxia during sleep. These findings suggest that peripheral chemosensitivity in patients with obstructive sleep apnoea syndrome may be decreased as a result of abnormality in dopaminergic mechanisms and that the reduced chemosensitivity observed in patients with obstructive sleep apnoea syndrome may affect the severity of hypoxia during sleep.

Adult↗
PubMed1999

Control of the dispersing process and pharmacokinetics in rats for lipid A analogue, E5531.

E5531 is a synthetic disaccharide analogue of lipid A which has a low toxicity but retains the ability to reduce production of tumour necrosis factor. This analogue has potential for use in the treatment of septic shock. An injectable formulation of E5531 would be useful, but dispersion in aqueous solution is a problem. In the present study the dispersing process for E5531 was evaluated using the pH-jump method (pH 11.0-->7.3). The size of the aggregates was decreased (reaching 20 nm) with increasing dispersing time in 0.003 M NaOH (pH 11.0). The membrane fluidity of the aggregates increased with increasing dispersing time. When prepared by the normal dilution method (pH 7.3-->7.3), the size of the aggregates remained constant at 140 nm and the membrane fluidity was smaller than that of samples prepared by the pH-jump method. This indicates that during dispersing at basic pH, the hydration proceeded in a normal manner, but then stopped, just after adjustment of the pH to 7.3. This suggests that the degree of hydration of the membrane is dependent on the dispersing time at pH 11.0. Using samples with different degrees of hydration and different membrane fluidity prepared by the pH-jump method, the pharmacokinetics and stability of the aggregates were evaluated after intravenous injection into rats. The data thus obtained confirmed that the membrane fluidity was correlated with the pharmacokinetics and stability in rat plasma. It was concluded that the pharmacokinetics of E5531 in rats can be controlled by changing the degree of hydration and membrane fluidity by means of using different dispersing times in alkaline solution (pH 11.0).

Animals↗
PubMed1999

Dipeptides containing L-arginine analogs: new isozyme-selective inhibitors of nitric oxide synthase.

Several L-arginine analogs are known as potent inhibitors of nitric oxide synthase (NOS). We recently synthesized dipeptides containing such amino acids, and found that they are potent and isozyme-selective NOS inhibitors. For example, S-methyl-L-isothiocitrullinyl-L-phenylalanine showed 66-fold selectivity for iNOS over nNOS, while S-methyl-L-isothiocitrullinyl-L-leucine and N(G)-nitro-L-argininyl-L-phenylalanine showed 20- and 14-fold selectivity, respectively. Interestingly, S-methyl-L-isothiocitrullinyl-D-phenylalanine showed no selectivity, and S-methyl-L-isothiocitrullinyl-L-phenylalanine showed competitive inhibition. These results suggest that each NOS isozyme has a cavity of different size near the C-terminal of the L-arginine binding site, and that the selectivity of inhibitors is due to the differences in the size of the cavity.

Animals↗
PubMed1999

Nonpeptide arginine vasopressin antagonists for both V1A and V2 receptors: synthesis and pharmacological properties of 4'-[5-(substituted methylidene)-2,3,4,5-tetrahydro-1H-1-ben zoazepine-1-carbonyl]benzanilide and 4'-[5-(substituted methyl)-2,3-dihydro-1H-1-benzoazepine-1- carbonyl]benzanilide derivatives.

Arginine vasopressin (AVP) has a dual action, i.e. vasoconstriction and water reabsorption via V1A and V2 receptors, and may play a role in a number of diseases, including congestive heart failure (CHF), hypertension, renal disease, edema, and hyponatremia. We have attempted to develop a new series of AVP antagonists for both V1A and V2 receptors based on the hypothesis that the blockade of both V1A and V2 receptors might be beneficial to CHF patients. In this report, a series of compounds structurally related to 4'-[5-(substituted methylidene)-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl ]benzanilide (exo-olefin isomer) and 4'-[5-(substituted methyl)-2,3-dihydro-1H-1-benzoazepine-1-carbonyl]benzanilide (endo-olefin isomer) were synthesized and examined to have AVP antagonist activity for both V1A and V2 receptors. As a result, it was found that the (E)-exo-olefin isomers showed more potent binding affinity compared with endo-olefin isomers. Among these (E)-exo-olefin isomers, (E)-N-methyl-{1-[4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahyd ro-1H-1 -benzoazepin-5-ylidene}acetamide (14) exhibited the most potent binding affinity and (E)-N-methyl-(1-{4-[2-(4-methylphenyl)benzoylamino]benzoyl}-2,3,4,5- tetrahydro-1H-1-benzoazepin-5-ylidene)acetamide (20) exhibited a high AVP antagonist activity for both V1A and V2 receptors after intravenous administration. Details of the synthesis and pharmacological properties of this series are presented.

Animals↗
PubMed1999

Cytoplasmic maturation for activation of pig follicular oocytes cultured and arrested at metaphase I.

A large population (62-90%) of pig follicular oocytes can mature to metaphase II after culture for 48 h. However, a proportion (6-22%) remain in an immature stage at metaphase I (metaphase I-arrested). The main objective of this study was to determine whether the cytoplasm of metaphase I-arrested pig oocytes is capable of being activated by sperm penetration or parthenogenetic stimulation. After culture for 48 h, oocytes without a polar body (73% were shown to be at metaphase I after staining) and those with a polar body (94% were at metaphase II) were fertilized in vitro. A total of 69% and 62% of the oocytes were activated to form a female pronucleus, respectively, and the rate of polar body extrusion induced by fertilization in the activated oocytes was 90% (the first polar body) and 95% (the second polar body), respectively. When oocytes without and with a polar body were stimulated with an electric pulse, 53% and 81% of the oocytes were activated, respectively. The rate of polar body extrusion in the activated oocytes was 73% (the first polar body) and 79% (the second polar body), respectively. In contrast, young metaphase I oocytes cultured for 24 h had low (6%) or zero activation rate after in vitro fertilization or electric pulse stimulation. However, about one-third of the young metaphase I oocytes penetrated by spermatozoa after in vitro fertilization responded to electric pulse 12 h after insemination, and almost all (93%) were activated when they were stimulated 24 h after insemination. Patterns of polypeptide synthesis and histone H1 kinase activity were similar in metaphase I-arrested and metaphase II oocytes, and were characterized by increase in a 25 kDa polypeptide and by decrease in kinase activity. Although the first step of meiotic division is impaired, these results indicate that metaphase I-arrested oocytes are mature cytoplasmically.

Analysis of Variance↗
PubMed1999

L-DOPS-Accelerated recovery of locomotor function in rats subjected to sensorimotor cortex ablation injury: pharmacobehavioral studies.

Central norepinephrine (NE) has been shown to play a beneficial role in amphetamine-facilitated recovery of behavior. To give insight into understanding the mechanism, the present studies were conducted to examine (a) the effects of L-threo-3,4-dihydroxyphenylserine (L-DOPS) combined with benserazide (BSZ; a peripheral aromatic amino acid decarboxylase inhibitor) and L-3,4-dihydroxyphenylalanine (L-DOPA), precursors of NE and dopamine (DA), respectively, on the recovery from beam-walking performance deficits in rats subjected to unilateral sensorimotor cortex ablation injury, and (b) the relationships between the behavioral recovery and the frequency of postoperative training and the size of ablation injury. It was found that the combined treatments with L-DOPS and BSZ promoted the recovery of locomotor function as early as 24 hours after injury. L-DOPA alone, however, did not facilitate behavioral recovery. The results of assay for the tissue levels of NE and its major metabolite (3-methoxy-4-hydoxyphenylethylene glycol; MHPG) in the brain using high-pressure liquid chromotography showed MHPG, but not NE, significantly increased in the cerebellum and the hippocampus. The behavioral recovery was also significantly correlated with the frequency of training subsequent to injury, but inversely with the size of cortex ablation. These results suggest that NE is likely to modulate functional recovery in this rodent model.

Animals↗
PubMed1999

Ganglion-cell tumor of the filum terminale: immunohistochemical characterization.

A case of an unusual spinal neuronal tumor is described in a 36-year-old woman presenting with a buttock pain. The spinal tumor was fully characterized by neuroradiological means, and in particular MRI was of significant value in delineating the extension of the tumor within the spinal canal and its exophitic growth pattern. Pathologically, a well circumscribed tumor originating from the intradural filum terminale characteristically comprised both large and small cells, resembling mature and immature neuronal cells, respectively. In addition, two neuronal markers, i.e., chromogranin A (CGA) and neuron-specific enolase (NSE), and other markers such as glial fibrilary acidic protein (GFAP), S-100 protein, HNK-1, tyrosine hydroxylase and beta 2-microgloblin were investigated immunohistochemically. We found that both neuronal cells expressed immunoreactivity for CGA and NSE, and small neuronal cells showed more intense CGA immunoreactivity, indicating an earlier stage of neuronal differentiation. Weakly positive immunoreactivity for HNK-1 was also demonstrated in small neuronal cells, consistent with evidence of maturation along a neuronal differentiation. From these findings a pathological diagnosis of ganglioneuroma was made. This unique group of ganglion-cell spinal tumors is reviewed in the literature and differential diagnosis and immunohistochemical features are discussed.

Adult↗
PubMed1999

Neurocysticercosis without detectable specific antibody.

A 19-year-old girl who had lived in India for five years until 1992 was admitted to Hokuto Hospital after general seizures which lasted for fifteen minutes. Cerebral magnetic resonance imaging (MRI) showed a ring-enhanced lesion of 6 mm in diameter in the right parietal lobe. She underwent surgical resection after diagnosis of the brain tumor. Histopathological examinations revealed that the resected tumor was a cysticercus of Taenia solium (T. solium), and we concluded that her seizures were caused by neurocysticercosis. Serological examinations by enzyme-linked immunosorbent assay (ELISA) and immunoblots to detect specific antibody against the glycoproteins of T. solium showed no detectable antibody response. The patient is under careful observation in our out-patient clinic with no medication.

Adult↗
PubMed1999

Co-aggregation as a virulent factor of Streptococcus sanguis isolated from infective endocarditis.

The pathogenicity of strains of the Streptococcus sanguis group, isolated from infective endcarditis, was studied by measuring the development of subcutaneous abscesses in mice after infection with S. sanguis and Actinomyces viscosus either singly or as co-aggregated pairs. The pathogenicity of the co-aggregates was also examined in various viable combinations of the two bacterial species. More abscesses were formed by A. viscosus than the S. sanguis group including clinical isolates. Abscess formation by co-aggregates of combinations of each isolate and A. viscosus produced a higher percentage of abscess formation than those caused by infection with a pure suspension of A. viscosus or tested streptococci. Co-aggregated cells were more resistant to phagocytosis and killing by neutrophils in vivo. These results indicated that S. sanguis group streptococci isolated from infective endocarditis are able to co-aggregate and resist phagocytosis. The ability of co-aggregation of S. sanguis may serve as a survival mechanism in a host defense system and may be linked with virulence of this bacteria.

Abscess↗
PubMed1999

Detection of anti-branched chain 2-oxo acid dehydrogenase complex (BCOADC)-E2 antibody in primary biliary cirrhosis by ELISA using recombinant fusion protein.

Anti-M2 of anti-mitochondrial antibody (AMA) is a serological marker of primary biliary cirrhosis (PBC). Anti-pyruvate dehydrogenase complex-E2 (anti-PDC-E2) is recognized as the most frequently occurring anti-M2, and a routine laboratory test for this antibody has already been established. However, it is also known that there are patients with PBC who are negative for anti-PDC-E2. For the serological diagnosis of these patients, immunoblotting for anti-M2s is indicated. However, the technique currently utilized is too laborious to allow testing of a large number of samples. In this study, we have developed an enzyme-linked immunosorbent assay (ELISA) using a recombinant fusion protein in order to evaluate anti-branched chain 2-oxo-acid dehydrogenase complex-E2 (anti-BCOADC-E2), another frequently occurring anti-M2 in PBC patients. KB cell lines (CCL 17) were utilized as source material, and BCOADC-E2 cDNA (971 bp) including the lipoic acid binding domain was amplified by polymerase chain reaction. The amplified region was subcloned into pEX-3 vectors and expressed, and the resulting fusion protein (beta-galactosidase/BCOADC-E2) was utilized as antigen for an ELISA. We ascertained the specificity of this antigen by inhibition tests with ELISA and immunoblotting. We defined the cut-off optical density (OD) value as the mean + 3 SD (0.146) of sera from 60 normal controls. Anti-BCOADC-E2 could not be detected with this assay in sera from normal controls and from patients with autoimmune hepatitis and chronic viral hepatitis. Anti-BCOADC-E2 was detected in 119 of 210 sera (56.7%) from patients with PBC. In addition, anti-BCOADC-E2 was detected in 48 of 99 (48.5%) sera from PBC patients who were negative for anti-PDC-E2. Here, we have succeeded in developing a new ELISA for detecting anti-BCOADC-E2. This system is antigen-specific and easily performed. This assay should allow routine testing of a large number of serum samples, and should become especially useful for the serodiagnosis of anti-PDC-E2-negative PBC patients.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗