Control of tunnel barriers in multi-wall carbon nanotubes using focused ion beam irradiation.

We have formed tunnel barriers in individual multi-wall carbon nanotubes using the Ga focused ion beam irradiation. The barrier height was estimated by the temperature dependence of the current (Arrhenius plot) and the current-voltage curves (Fowler-Nordheim plot). It is shown that the barrier height has a strong correlation with the barrier resistance that is controlled by the dose. Possible origins for the variation in observed barrier characteristics are discussed. Finally, the single electron transistor with two barriers is demonstrated.

Acute non-hemolytic transfusion reactions and HLA class I antibody: advantages of solid phase assay compared with conventional complement-dependent assay.

To evaluate the specific reactivity of HLA Class I antibodies (HLA-I Abs) in acute non-hemolytic transfusion reactions (ANHTRs) using solid phase assays (SPAs) and conventional complement-dependent lymphocyte cytotoxicity test (LCT). ANHTRs are major issues in transfusion medicine. Anti-leukocyte antibodies have been implicated as one of the causative agents of transfusion-related acute lung injury (TRALI) and febrile reaction. Antibodies to HLA Class I and/or Class II (HLA Abs) have been intensively studied using SPAs for TRALI, but not for febrile reaction. About 107 patients and 186 donors associated with ANHTRs were screened for HLA Abs by SPAs such as enzyme-linked immunosorbent assay (ELISA) and the Luminex method. When HLA-I Ab was detected, its specific reactivity was evaluated by comparing its specificity identified by the Luminex method using recombinant HLA molecules and cognate HLA antigens (Ags), as well as LCT with or without anti-human globulin (AHG). The incidences of HLA Abs were as high as 32.7% of patients' serum samples and 16% of donors' serum samples. The incidence of HLA-I Abs did not differ significantly between cases of febrile and allergic reactions. However, HLA-I Abs associated with febrile reaction showed a significantly higher rate of possessing specific reactivity to cognate HLA Ags than those associated with allergic reactions. In addition, the Luminex method enabled the detection of HLA-I Abs much earlier than AHG-LCT in serum samples from a patient with febrile reaction and platelet transfusion refractoriness (PTR). SPAs seem more useful than AHG-LCT for evaluating reactivity of antibodies in ANHTR cases.

Investigation of Physiological Swelling on Conductivity Distribution in Lower Leg Subcutaneous Tissue by Electrical Impedance Tomography.

There is a strong need for a non-invasive measurement technique that is capable of accurately identifying the physiological condition change or heterogeneity of subcutaneous adipose tissue (SAT) by localizing the abnormalities within the compartment. This paper aims to investigate the feasibility of Electrical Impedance Tomography (EIT) to assess the interstitial fluid in subcutaneous adipose tissue as an enhancement method of bioelectrical impedance spectroscopy (BIS). Here, we demonstrate the preliminary result of EIT with a wearable 16 electrodes sensor. The image-based reference EIT with fat weighted threshold method is proposed. In order to evaluate the performance of our novel method, a physiological swelling experiment is conducted, and Multi-Frequency Bioelectrical Impedance Analysis (MFBIA) is also applied as a comparison with EIT results. The experimental results showed that the proposed method was able to distinguish the physiological swelling condition and effectively to remove the unexpected background noise. Furthermore, the conductivity variation in the subcutaneous layer had a good correlation with extracellular water volume change from MFBIA data; the correlation coefficient R2 = 0.927. It is concluded that the proposed method provides a significant prospect for SAT assessment.

Pituitary-directed leukemia inhibitory factor transgene forms Rathke's cleft cysts and impairs adult pituitary function. A model for human pituitary Rathke's cysts.

Leukemia inhibitory factor (LIF) and LIF receptors are expressed in adenohypophyseal cells and LIF regulates pituitary hormone transcription and cell replication in vitro. Therefore, transgenic mice expressing pituitary-directed LIF driven by the rat growth hormone (GH) promoter were generated to evaluate the impact of LIF on pituitary development. Three founders were established with diminished linear growth and body weight (57-65% of wild type [WT]), and intense anterior pituitary LIF immunoreactivity. Cystic cavities observed in pituitary anterior lobes were lined by cuboidal, ciliated epithelial cells, focally immunopositive for cytokeratin and S-100 protein and immunonegative for adenohypophyseal hormones. Transgenic pituitaries showed decreased GH (40%) and prolactin (PRL) (26%) cells, and decreased GH and PRL mRNAs by in situ hybridization. ACTH cells increased 2.2-fold, whereas gonadotrophs and thyrotrophs were unchanged. Serum GH was undetectable (< 0.78 ng/ml), PRL levels were one third of WT (P < 0.05), IGF-I levels were 30% of WT (P < 0. 001), and T4 was normal. 10 human pituitary Rathke's cysts studied all showed conclusive LIF immunoreactivity in cyst-lining cells. Thus, intrapituitary murine LIF overexpression causes cystic invaginations from the anterior wall of Rathke's cleft, suggesting failed differentiation of Rathke's epithelium to hormone-secreting cells. Arrested murine pituitary maturation with formation of pituitary Rathke's cleft cysts, GH deficiency, and short stature provide a model to study human Rathke's cyst pathogenesis.

Human and murine pituitary expression of leukemia inhibitory factor. Novel intrapituitary regulation of adrenocorticotropin hormone synthesis and secretion.

Leukemia inhibitory factor (LIF) gene expression was detected in human fetal pituitary tissue by expression of LIF mRNA transcripts, protein immunocytochemistry, and immunoelectron microscopy. Fetal LIF immunoreactivity colocalized with 30% of ACTH-expressing cells, approximately 20% of somatotrophs, and approximately 15% of non-hormone-expressing cells. LIF was also strongly expressed in normal adult pituitary and in four growth hormone-producing and two ACTH-producing adenomas, but not in eight nonfunctioning pituitary tumors. Culture of fetal cells expressing surface LIF-binding sites demonstrated predominance of in vitro ACTH secretion as compared with other pituitary hormones. In AtT-20 murine cells, LIF (ED50 10 pM) stimulated basal proopiomelanocortin mRNA levels by 40% and corticotropin-releasing hormone-induced ACTH secretion (two- to threefold), as did oncostatin M (ED50 30 pM), a related peptide. ACTH responses were not further enhanced by both cytokines together, which is consistent with their shared receptor. Anti-LIF antiserum neutralized basal and LIF-induced ACTH secretion, suggesting autocrine regulation of ACTH by LIF. The results show that human pituitary cells express the LIF gene and LIF-binding sites, predominantly in corticotrophs. Pituitary LIF expression and LIF regulation of proopiomelanocortin and ACTH reflect an intrapituitary role for LIF in modulating early embryonic determination of specific human pituitary cells and as a paracrine or autocrine regulator of mature ACTH.

Copolymerisation of ethylene with polar monomers by using palladium catalysts bearing an N-heterocyclic carbene-phosphine oxide bidentate ligand.

We report the synthesis and characterisation of palladium complexes bearing an N-heterocyclic carbene-phosphine oxide bidentate ligand and their use as catalysts for ethylene polymerisation and ethylene/polar monomer copolymerisation.

Combined injuries of the brachial plexus and spinal cord.

A total of 11 patients with combined traumatic injuries of the brachial plexus and spinal cord were reviewed retrospectively. Brachial plexus paralysis in such dual injuries tends to be diagnosed and treated late and the prognosis is usually poor. The associated injuries, which were all on the same side as the plexus lesion, were to the head (nine cases), shoulder girdle (five), thorax (nine) and upper limb (seven). These other injuries were responsible for the delayed diagnosis of brachial plexus paralysis and the poor prognosis was probably because of the delay in starting treatment and the severity of the associated injuries. When such injuries are detected in patients with spinal cord trauma, it is important to consider the possibility of involvement of the brachial plexus.

Volumetric breast density is essential for predicting cosmetic outcome at the late stage after breast-conserving surgery.

BACKGROUND: The critical issue related to breast-conserving therapy (BCT) is that cosmetic outcomes deteriorate with long-term follow-up. There is little research for breast density as a predictor of cosmetic outcomes at the late stage after BCT. To improve the long-term quality of life after BCT of breast cancer patients, the correlation of volumetric breast density (VBD) and cosmetic outcome at the late stage after BCT was evaluated. STUDY DESIGN: Breast volume, fibroglandular tissue volume, adipose tissue volume, and VBD were calculated on mammography using image analysis software (Volpara(®)) in 151 patients with BCT. Furthermore, the correlation of breast density and the change of breast volume over time was analyzed on mammography in 99 patients who were followed-up long-term after BCT. RESULTS: On multivariate analysis, VBD was a predictor of cosmetic outcome after BCT with percent breast volume excised (PBVE). Decreased adipose tissue volume and increased fibrosis were more common in patients with VBD < 15%. Furthermore, remnant breast volume continued to decrease over time in low breast density patients during long-term follow-up. 93% of patients with VBD ≥ 15% and PBVE < 10% had a better cosmetic outcome, while 60% of patients with VBD < 15% and PBVE ≥ 10% had a worse cosmetic outcome after BCT. CONCLUSIONS: While PBVE was involved in cosmetic outcome at the early stage after BCT, VBD was associated with cosmetic outcome at the late stage after BCT. Thus, a combination of VBD and PBVE could predict cosmetic outcome after BCT and contribute to the selection for the appropriate BCT.

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Telomerase plays a key role in the maintenance of chromosomal stability in tumors, but the mechanism regulating telomerase activity is still unclear. Recent studies have suggested that c-myc may be vital for regulation of hTERT mRNA expression and telomerase activity. In this study, we investigated the changes of telomerase activity and telomerase-related genes induced by herbimycin A in K562 human chronic myelogeous leukemic cells. Telomerase activity showed a biphasic pattern in herbimycin A-treated K562 cells. Initially, the telomerase activity decreased along with the decline of cells in S and G2/M phases, but it recovered slightly at the end of treatment. Expression of mRNA for the telomerase catalytic subunit (hTERT) was decreased before the decline of telomerase activity, and increased slightly before the reactivation of telomerase activity. During herbimycin A treatment, both c-myc and cyclin D1 mRNA showed transient downregulation before the increase of G1 cells. Herbimycin A treatment caused the downregulation of both telomerase activity and hTERT mRNA in cyclin D1-transfected K562 cells, while telomerase activity was partially restored in c-Myc-transfected cells. In contrast, hTERT-transfected K562 cells maintained a high level of telomerase activity during herbimycin A treatment. Neither the template RNA component of telomerase (hTERC) nor telomerase-associated protein (TEP-1) were altered in any of the transfected K562 cells. These results indicate that telomerase activity is mainly regulated by hTERT, and that c-Myc protein is one of the positive regulators of hTERT in leukemic cells but is not enough to counteract the downregulation of telomerase activity by herbimycin A completely.

Hydrometallurgical recovery of zinc from ashes of automobile tire wastes.

Study has been performed on the investigation of metal leaching behavior for fly and bottom ashes from automobile tire wastes using acid and alkaline solutions from both viewpoints of environmental protection and resource utilization. The two ashes were found to contain substantial amounts of zinc and iron along with small quantities of cobalt, manganese, magnesium, copper, titanium and aluminum. The fly ash contained a much larger amount of zinc than the bottom ash, and seems to be a promising secondary source for the metal. Effects of such experimental parameters as temperature, time and solid-liquid ratio on the leaching behavior were investigated. Using three mineral acids and citric acid, selective leaching of zinc was successfully attained; the concentration of zinc in the leach liquors from the fly ash reached as high as 20 g l(-1) while the iron leaching was much suppressed. Selective separation of zinc was also attained in the leaching with alkaline solutions, though the percent leaching was lower than that in the acid leaching. Moreover, solvent extraction and precipitation were applied to the metal-loaded leach liquors as downstream processing to evaluate the feasibility of zinc recovery.

Ectopic expression of c-myc fails to overcome downregulation of telomerase activity induced by herbimycin A, but ectopic hTERT expression overcomes it.

Correction to: Leukemia (2000); 14: 1260­1265; doi: 10.1038/sj.leu.2401828. Since the publication of the above article the authors have identified an error in Figure 1. Figure 1 shows the modulation of telomerase activity by herbimycin A in K562 cells: (a) cell cycle and (b) telomerase activity, mRNA expressions of hTERT, hTERC, TEP-1, c-myc, cyclin D1 and b-actin, and c-Myc protein. The authors however wish to inform the readers that Figure 1b incorrectly shows hTERT mRNA, which is the result of herbimycin A treatment of cyclin-D1-transfected K562 cells (Figure 3b, hTERT mRNA). While preparing Figure 1, the authors mistakenly submitted a figure that used the incorrect photo data following confusion regarding file names. The correct figure can be found below: The authors wish to apologise for any inconvenience caused and confirm that the conclusions drawn from this research are not affected by this error.

Disrupted murine leukemia inhibitory factor (LIF) gene attenuates adrenocorticotropic hormone (ACTH) secretion.

Leukemia Inhibitory Factor (LIF) and its receptors in human and mouse pituicytes are expressed abundantly in corticotrophs and somatotrophs. As LIF induces POMC transcription and LPS-mediated stress also induces hypothalamic and pituitary LIF expression, we studied ACTH secretion in LIF knockout (LIF KO) mice. Basal ACTH levels were lower in LIF KO mice (p<0.05) and after 36 hours fasting, LIF KO mice had lower ACTH levels (38% of WT littermates, p=0.014 ). Delivery of LIF (1.2 microg/day) via implantation of subcutaneous osmotic pumps restored ACTH (p=0.006 vs PBS replacement) and corticosterone (p=0.02 vs PBS replacement) levels within three days. After five days, pumps were removed and two days later, ACTH levels had reverted to pre-treatment values. In contrast, GH concentrations were attenuated by LIF replacement to LIF KO mice. Thus, absence of LIF in LIF KO mice results in attenuated ACTH levels indicating that LIF plays an important intrapituitary role in HPA axis development and regulation.

Leukemia inhibitory factor (LIF) induces acute adrenocorticotrophic hormone (ACTH) secretion in fetal rhesus macaque primates: a novel dynamic test of pituitary function.

Leukemia Inhibitory Factor (LIF), a pituitary cytokine, and LIF receptors are expressed in human fetal and adult adenohypophyseal cells. LIF induces adrenocorticotropin hormone (ACTH) secretion in vitro and potently synergizes with both corticotropin-releasing hormone (CRH) and cAMP-induced pro-opiomelanocortin (POMC) transcription. We therefore investigated the effects of intra-carotid administration of recombinant human LIF to chronically catheterized fetal non-human primates. LIF induced fetal monkey ACTH secretion in a time- and dose dependent manner. Maximal ACTH induction (12-fold) was achieved with 100 micrograms/kg after 60 minutes (p < 0.01). CRH (10 micrograms/kg) also induced ACTH secretion 4.8-fold at 60 minutes. Co-injection of LIF (50 micrograms/kg) and CRH (10 micrograms/kg) synergistically induced ACTH levels in a time-dependent manner up to 23-fold after 60 minutes. Thus, LIF alone induces ACTH secretion and LIF acts in synergy with CRH in vivo. As LIF is expressed early in human fetal pituitary development, and potentiates corticotroph function both in vitro and in vivo, this immuno-regulatory cytokine may be useful for clinical testing of the hypothalamic-pituitary-adrenal axis.

Leukemia inhibitory factor gene improves skin allograft survival in the mouse model.

BACKGROUND: Leukemia inhibitory factor (LIF) is a widely expressed cytokine involved in both local and systemic immune response. Furthermore, it has been implicated in various immunological processes including thymic T cell maturation and embryo implantation. We investigated implication of various modalities in the application of prolonged and viable allograft to the wound, using cytokines and growth factors. MATERIALS: BALB/c and B6D2F1 strains of mice were used either as a skin graft donor or host. LIF cDNA inserted in plasmid vector or the vector alone was injected intradermally in graft skin and observed up to 21 days. LIF, LIF-receptor, gp130, as well as type 1 and 2 T helper cytokine expressions were investigated by reverse transcription polymerasse chain reaction, in situ hybridization, and histological studies. RESULTS: LIF cDNA-treated groups showed significantly improved graft survival compared to the vector-treated control in 21 days postoperatively for grafting from B6D2F1 to BALB/c and BALB/c to B6D2F1. LIF and LIF receptor mRNA expressions were observed 24 hr and 21 days posttransplantation. The gp130 expression was only observed in LIF-treated B6D2F1 to BALB/c allografting on day 21 posttransplantation. LIF transcripts were strongly present in the epidermal, dermal, and subdermal tissues as determined by an in situ hybridization of LIF-treated grafting. CONCLUSIONS: These results suggest that LIF cDNA treatment is an effective and beneficial adjuvant for the skin allograft survival. Improved skin allograft modulation by cytokine gene transfer is a potentially promising therapy for temporary large skin coverage.

Clinical significance of reverse redistribution on 24-hour delayed imaging of exercise thallium-201 myocardial SPECT: comparison with myocardial fluorine-18-FDG-PET imaging and left ventricular wall motion.

UNLABELLED: Clinical significance of reverse redistribution on 24-hr delayed images after exercise 201Tl myocardial SPECT was investigated in 16 patients with recent myocardial infarction. METHODS: Findings of 24-hr delayed 201Tl SPECT imaging were compared with those of glucose-loaded 18F-fluorodeoxyglucose (FDG) imaging by myocardial PET and with left ventricular wall motion obtained by bi-plane contrast left ventriculography. In each patient, transaxial thallium images and corresponding 18F-FDG images were divided into five ROIs. RESULTS: Reverse redistribution was found in 15 of 80 regions. The mean FDG activity score in regions with reverse redistribution was significantly lower than that in regions having normal or slightly decreased thallium activity on 24-hr delayed imaging; it was significantly higher than that in regions having severely decreased or no thallium activity on 24-hr delayed imaging. The mean wall motion score in regions with reverse redistribution was significantly lower than in regions with normal or slightly decreased thallium activity, however, it was significantly higher than that in regions with moderately or more decreased thallium activity. CONCLUSION: These findings demonstrate that in regions showing reverse redistribution on 24-hr delayed 201Tl imaging, myocardial exogenous glucose utilization and left ventricular wall motion had deteriorated, but were not on a level with the scar.

Evaluation of left ventricular early diastolic performance by color tissue Doppler imaging of the mitral annulus.

A noninvasive assessment of left ventricular (LV) diastolic performance by tissue Doppler imaging was performed in 56 patients (8 patients with atypical chest pain, 42 with coronary artery disease with a previous myocardial infarction, and 6 without a previous myocardial infarction) who underwent cardiac catheterization. Mitral annular velocity (MAV) during early ventricular diastole was obtained by M-mode color tissue Doppler imaging at the posterior corner of the mitral annulus. In each patient, the negative peak of the first derivative of LV pressure decay (peak -dP/dt) and a time constant of LV relaxation (tau) were calculated from the LV pressure waves obtained by a catheter-tip micromanometer. LV end-systolic volume index was measured from contrast left ventriculography. MAV during early diastole was significantly correlated with tau (r = -0.73, p <0.001), peak -dP/dt (r = 0.58, p <0.001), and LV end-systolic volume index (r = -0.63, p <0.001). On multivariate regression analysis with MAV during early diastole, tau and LV end-systolic volume index were selected as prime determinants (r = 0.80, p <0.001). These findings suggest that MAV during early diastole has a direct relation to LV elastic recoil as well as to LV relaxation. MAV during early diastole gives important information regarding LV behavior in late systole to early diastole where LV early diastolic performance is determined.

Striking effect of left ventricular systolic performance on propagation velocity of left ventricular early diastolic filling flow.

Propagation velocity of left ventricular (LV) early diastolic filling flow (PVE) has been acknowledged as a useful parameter for LV early diastolic performance; however, the effect of LV systolic performance on PVE is not fully understood. Thus the purpose of this study was to investigate such an effect. Propagation of LV early diastolic filling flow was visualized by M-mode color Doppler imaging, and the slopes of the peak velocity tracings were measured as PVE in 150 patients who underwent coronary angiography. In cardiac catheterization, mean pulmonary capillary wedge pressure, time constant tau of LV pressure decay, LV end-systolic volume index, and LV ejection fraction were obtained. In univariate regression analysis, PVE significantly correlated with LV end-systolic volume index (r = -0.68, P <.001), LV ejection fraction (r = 0.66, P <.001), and time constant tau (r = -0.52, P <.001). In multivariate regression analysis, PVE was regressed by the LV end-systolic volume index, tau, and mean pulmonary capillary wedge pressure. The contribution of each parameter to the variance of the PVE was 46%, 3%, and 2%, respectively. A break-point linear regression analysis showed that the relation between the LV end-systolic volume index and PVE was much better characterized by a broken line than a straight line. The broken line had a steeper slope in patients with LV end-systolic volume index < or =41 mL/m(2) than in those with >41 mL/m(2). These findings suggest that PVE is determined mainly by LV systolic performance and partly by both LV relaxation and LV filling pressure. Left ventricular systolic performance may play a key role in generating a much faster PVE, especially in patients with relatively better LV systolic performance.

Left ventricular isovolumic relaxation flow and left ventricular systolic performance.

We investigated isovolumic relaxation flow in patients with coronary artery disease (CAD) and evaluated the relationship between its velocity and left ventricular performance in 23 patients with atypical chest pain, 30 patients with CAD without prior myocardial infarction (MI), and 57 patients with prior MI, in whom cardiac catheterization was performed. The isovolumic relaxation flow velocity was measured at the basal portion of the left ventricle with pulsed Doppler echocardiography. The isovolumic relaxation flow ( > 15 cm/sec) was detected in 98 of 110 patients. The isovolumic relaxation flow velocity was significantly lower in patients with prior MI than in patients with atypical chest pain (p < 0.001) and in those with CAD without prior MI (P < 0.05). It was significantly lower in patients with CAD without prior MI than in those with atypical chest pain (p < 0.05). The isovolumic relaxation flow velocity showed a significant positive correlation with left ventricular ejection fraction. It also showed a significant negative correlation with left ventricular end-systolic volume index. These findings suggest that the isovolumic relaxation flow velocity is decreased in patients with CAD and is influenced by left ventricular systolic performance. Isovolumic relaxation flow may be a clinical manifestation of elastic recoil of the left ventricle.

Differentiation of abnormal relaxation pattern with aging from abnormal relaxation pattern with coronary artery disease in transmitral flow with the use of tissue Doppler imaging of the mitral annulus.

An abnormal relaxation pattern in transmitral flow velocity waveforms has been observed in older healthy subjects as well as in patients with heart disease. Accordingly, we investigated whether the hemodynamic differences between patients with coronary artery disease (CAD) with an abnormal relaxation pattern in transmitral flow (ratio of E-wave to A-wave velocities < 1.0) and healthy older subjects with an abnormal relaxation pattern can be distinguished with the use of mitral annular velocity (MAV) during early diastole. We measured MAV in the longitudinal direction of the heart during early diastole by M-mode color tissue Doppler imaging in 24 patients with atypical chest pain (defined as healthy subjects in this study) and 70 patients with CAD who underwent cardiac catheterization. In all patients a time constant of left ventricular pressure decay (tau) and the left ventricular (LV) end-systolic volume index were also measured. Twenty-one healthy subjects and 59 patients with CAD had an abnormal relaxation pattern in their transmitral flow. The age, heart rate, mean blood pressure, and ratio of E-wave to A-wave velocities were not different between the two groups. However, the tau was longer and the LV end-systolic volume index was greater in patients who had an abnormal relaxation pattern with CAD than in healthy subjects with an abnormal relaxation pattern. The MAV during early diastole was lower in the former than in the latter (5.8 +/- 1. 9 vs 9.8 +/- 1.9 cm/s, P <.001). Mitral annular velocity during early diastole by M-mode color tissue Doppler imaging can detect the differences in LV relaxation and LV systolic performance between the abnormal relaxation pattern with CAD and the physiologically abnormal relaxation pattern with aging, providing further information regarding the meaning of an LV abnormal relaxation pattern.

The gene for alternative oxidase-2 (AOX2) from Arabidopsis thaliana consists of five exons unlike other AOX genes and is transcribed at an early stage during germination.

We investigated the expressions of genes for alternative oxidase (AOX1a, AOX1b, AOX1c and AOX2) and genes for cytochrome c oxidase (COX5b and COX6b) during germination of Arabidopsis thaliana, and examined oxygen uptakes of the alternative respiration and the cytochrome respiration in imbibed Arabidopsis seeds. A Northern blot analysis showed that AOX2 mRNA has already accumulated in dry seeds and subsequently decreased, whereas accumulation ofAOX1a mRNA was less abundant from 0 hours to 48 hours after imbibition and then increased. The increase of the capacity of the alternative pathway appeared to be dependent on the expressions of both AOX2 and AOX1a. On the other hand, steady-state mRNA levels of COX5b and COX6b were gradually increased during germination, and the capacity of the cytochrome pathway was correlated with the increase of expressions of the COX genes. Antimycin A, the respiratory inhibitor, strongly increased the expression of AOX1a but had no effect on the expression of AOX2. A 5'RACE analysis showed that AOX2 consists of five exons, which is different from the case of most AOX genes identified so far. Analysis of subcellular localization of AOX2 using green fluorescent protein indicated that the AOX2 protein is imported into the mitochondria.