Polymorphisms in genes involved in the free-radical process in patients with sudden sensorineural hearing loss and Ménière's disease.

The etiologies of idiopathic sudden sensorineural hearing loss (SSNHL) and Ménière's disease remain unclear. Recently, accumulating evidence has demonstrated that free radicals are related to the pathology of inner ear disease. Because genetic factors may contribute partly to the etiologies of SSNHL and Ménière's disease, we investigated the association between genetic polymorphisms located in genes related to the free-radical process and susceptibility to SSNHL and Ménière's disease. We compared 83 patients affected by SSNHL and 83 patients affected by Ménière's disease with 2048 adults (for SSNHL) and 1946 adults (for Ménière's disease) who participated in the National Institute for Longevity Sciences, Longitudinal Study of Aging. Multiple logistic regression was used to calculate odds ratios (ORs) for SSNHL and Ménière's disease in individuals with polymorphisms in the genes: methionine synthase (MTR; rs1805087); methionine-synthase reductase (MTRR; rs1801394); nitric oxide synthase 3 (NOS3; rs1799983); caveolin 1 (Cav1; rs3840634); melatonin receptor 1B (MTNR1B; rs1387153); NAD(P)H oxidase p22(phox) subunit (NADH/NADPHp22phox; rs4673); and mitochondria 5178 (MT5178; rs28357984). The NOS3 polymorphism was significantly associated with a risk of SSNHL; in addition, the OR for the NOS3 polymorphism and SSNHL risk was 2.108 (CI, 1.343-3.309) with adjustment for age and sex. The Cav1 polymorphism was significantly associated with a risk of Ménière's disease; moreover, the OR for the Cav1 polymorphism and Ménière's disease risk was 1.849 (CI, 1.033-3.310) with adjustment for age and sex. In conclusion, the NOS3 and Cav1 polymorphisms were significantly associated with the risk of SSNHL and Ménière's disease, respectively.

Comparison of contrast effect on the cochlear perilymph after intratympanic and intravenous gadolinium injection.

BACKGROUND AND PURPOSE: 3D-FLAIR imaging 24 hours after intratympanic gadolinium injection (IT-method) or 4 hours after IV injection (IV-method) has been used to visualize the endolymphatic hydrops in Ménière disease. The purpose of this study was to compare the degree of perilymph enhancement with the 2 methods and the perilymph contrast-effect difference with the IV-method in both sides in patients with unilateral Ménière disease. MATERIALS AND METHODS: Sixty-one patients with Ménière disease or sudden SNHL were included in this study. Thirty-nine patients who underwent the unilateral IT-method (Gd-DTPA was diluted 8-fold with saline) and 22 patients who underwent the IV-method (a double-dose of Gd-HP-DO3A; 0.4 mL/kg body weight [ie, 0.2 mmol/kg body weight]) at 3T were analyzed retrospectively. Regions of interest of the cochlear perilymph and the medulla oblongata were determined on each image, and the signal-intensity ratio between the 2 (CM ratio) was subsequently evaluated. The differences in the CM ratio between the 2 methods (Student t test) and the IV-method CM ratio between the affected and unaffected sides in patients with unilateral Ménière disease (paired t test) were evaluated. RESULTS: The IT-method CM ratio (2.98 ± 1.15, n = 39) was higher than the IV-method CM ratio (1.61 ± 0.60, n = 44; P < .001). In patients with unilateral Ménière disease who underwent the IV-method (n = 9), the CM ratio of the affected side (1.86 ± 0.74) was higher than that of the unaffected side (1.29 ± 0.31, P < .05). CONCLUSIONS: In general, the IT-method provides higher perilymph enhancement than the IV-method. In the patients with unilateral Ménière disease who underwent the IV-method, the affected side had a higher contrast effect.

Association of interleukin-1 gene polymorphisms with sudden sensorineural hearing loss and Ménière's disease.

Sudden sensorineural hearing loss (SSNHL) and Ménière's disease are the most common inner ear diseases in which the causes are unknown. As recent magnetic resonance imaging has demonstrated disruption of the blood-labyrinth barrier in these inner ear diseases, inflammatory reaction associated with increased permeability of the blood vessels may be involved. The genotypes of interleukin 1A (IL1A) (-889C/T; rs1800587) and interleukin 1B (IL1B) (-511C/T; rs16944) were determined using an allele-specific primer-polymerase chain reaction method in 72 patients with SSNHL, 68 patients with Ménière's disease, and 2202 control subjects living almost in the same area as the patients. A significantly higher prevalence of the IL1A-889T allele was observed in SSNHL and Ménière's disease compared with controls, although no significant difference in distribution of IL1B-511C/T genotypes was observed between the patients and controls. Adjusted odd ratios for SSNHL and Ménière's disease risks in the -889TT genotypes were 25.89 (95% confidence interval (CI) 12.19-54.98) and 18.20 (95% CI 7.80-42.46), respectively, after age and gender were taken as moderator variables. Our results suggested that IL1A is closely associated with susceptibility of SSNHL and Ménière's disease.

Morphological features of elderly patients with obstructive sleep apnoea syndrome: a prospective controlled, comparative cohort study.

OBJECTIVES: To investigate the pharyngeal morphologic features and its pathogenic role on obstructive sleep apnoea syndrome in the elderly population. DESIGN: Prospective controlled, comparative cohort study. SETTING: Territory referral centre. PARTICIPANTS: We enroled 320 consecutive patients with complaints of snoring who visited Nagoya University Hospital from January 2004 to December 2007. We also collected 26 control subjects aged over 60 years from community-dwelling people. MAIN OUTCOME MEASURES: We underwent a morphological evaluation, measurement of nasal resistance, assessment of daytime sleepiness and nocturnal polysomnography. RESULTS AND CONCLUSIONS: Two hundred and ninety-two patients were analysed. The constitution ratio of men, the body mass index and Epworth sleepiness scale were decreased with ageing. Tonsil size was reduced progressively with ageing. Retroglossal space was wider, and soft palate was lower in ≥ 60 year group than in < 40 year group. Retroglossal space was wide in elderly patients with sleep apnoea compared with control subjects. Tonsil size was not correlated to apnoea/hypopnoea index in ≥ 60 year group unlike the other generations. Modified Mallampati Score and tongue size were significantly but mildly correlated only in ≥ 60 year group. Width of fauces was correlated in all the groups. Multiple regression analysis showed that body mass index, age, gender, tonsil size and width of fauces were independent factors for apnoea/hypopnoea index. CONCLUSIONS: Morphologically, the tonsil could play a minor role but the width of fauces could play relatively a major role. Additionally, wide retroglossal space, low positional soft palate and large tongue size may be characteristics for elderly patients of obstructive sleep apnoea syndrome.

Anatomy-based surgery to remove thyroglossal duct cyst: two anomalous cases.

OBJECTIVE: To review previous reports and to discuss the management of branching polycystic and giant thyroglossal duct cysts. CASE REPORT: We present two cases of thyroglossal duct cyst: one a branching, polycystic thyroglossal duct cyst in an 11-year-old boy, and the other a giant thyroglossal cyst in a 41-year-old man. Such cysts are rare. Both patients were operated upon according to the methods of Sistrunk and Horisawa, and both had a satisfactory post-operative course. DISCUSSION: We discuss the most important aspects of such cyst removal procedures. CONCLUSION: Our experience suggests that surgery to remove an anomalous thyroglossal duct cyst should be performed using a technique based on the anatomy of the hyoid bone region.

NADH-generating substrates reduce peroxyl radical toxicity in RL-34 cells.

There is general agreement that oxidative stress may induce apoptotic and necrotic cell death. Recently it has been shown that NADH can be considered an important antioxidant as it reacts with peroxyl and alkoxyl radicals under in vitro conditions. Therefore, in the present study we hypothesized that an increase in intracellular NADH using specific substrates will protect RL-34 cells against cytotoxicity of 2'-azobis (2-amidinopropane) dihydrochloride (AAPH), which is a peroxyl radical generating compound. Cells treated for 24 hours with 6.0 mM AAPH were severely damaged: mitochondria were vacuolated, and the level of free radicals significantly increased. Both apoptotic and necrotic cells were detected (11.1% and 11.4%, respectively) even after 5 hours of treatment. Pretreatment of the cells with substrates which increase the intracellular level of NADH, such as lactate, beta-hydroxybutyrate, and ethanol, distinctly inhibited AAPH-induced reactive oxygen species (ROS) formation and cell death. On the other hand, acetoacetate (AcA), which decrease the intracellular level of NADH, had opposite effects. Interestingly, NADH-generating substrates augment, while AcA reduced superoxide radical formation induced by AAPH. These results may suggest that although NADH generating substrates may exert some deleterious effects within a cell by inducing reductive stress, they diminish alkoxyl or peroxyl radical cytotoxicity. The protection is associated with a decrease in ROS formation measured by dichlorofluorescein, but with an increase in superoxide radical formation.

Vestibular aqueduct in sudden sensorineural hearing loss.

OBJECTIVE: To evaluate the vestibular aqueduct in patients with sudden sensorineural hearing loss. METHODS: We evaluated 19 patients (12 men and seven women; age range, 22-79 years) with unilateral sudden sensorineural hearing loss, using computed tomography and magnetic resonance imaging. All these patients had unilateral sudden sensorineural hearing loss. We also evaluated 47 control subjects (22 men and 25 women; age range, 22-79 years). RESULTS: In sensorineural hearing loss affected ears, the width of the vestibular aqueduct at the midpoint and at the operculum was significantly greater than that in contralateral ears or in control ears. The width of the vestibular aqueduct at the midpoint and the operculum did not correlate with the audiometric threshold or the audiogram configuration. Contrast enhancement of the ipsilateral endolymphatic sac was observed in 17 of 19 patients with sudden sensorineural hearing loss (89 per cent). Eleven of these 17 patients also showed enhancement on the contralateral side, but no patient showed enhancement only on the contralateral side. In sensorineural hearing loss affected ears, the width of the vestibular aqueduct did not differ significantly between those patients with and without enhancement. CONCLUSIONS: The vestibular aqueducts of sudden sensorineural hearing loss affected ears are wider than those of controls. Precise imaging and evaluation of the inner ear is essential when investigating the pathological conditions responsible for sudden sensorineural hearing loss.

Blood flow to the promontory in cochlear otosclerosis.

OBJECTIVE: To investigate Schwartze sign with measurements of blood flow to the promontory in patients with cochlear otosclerosis. DESIGN: Prospective clinical study. SETTING: Tertiary referral centre. PARTICIPANTS: Five patients with cochlear otosclerosis and five control subjects. Significant decalcification around the cochlea was observed by computed tomography (CT) in patients with cochlear otosclerosis. However, no recognizable lesion was observed at the oval window in two patients. One patient had mixed hearing loss and four patients had sensorineural hearing loss without an air-bone gap. MAIN OUTCOME MEASURES: The relationship between CT findings and the presence or absence of Schwartze sign was investigated. Blood flow to the promontory was measured through the tympanic membrane using laser speckle flowgraphy and laser Doppler flowmetry. RESULTS: The Schwartze sign correlated significantly with otosclerotic lesions invading the promontory. Patients with otosclerosis exhibited elevated and pulsating blood flow to the promontory with the Schwartze sign. CONCLUSIONS: Computed tomography demonstrated that cochlear otosclerosis can exist without the oval window lesion. Schwartze sign can be used as a sign of the otosclerotic invasion to the promontory. The reddening of the Schwartze sign is likely due to increased blood flow.

A gene for a Class II DNA photolyase from Oryza sativa: cloning of the cDNA by dilution-amplification.

Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine [6-4] pyrimidone photoproduct (6-4 product). Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. Two classes of photolyases (class I and class II) repair CPDs. A gene that encodes a protein with class II CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii. We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification. The cDNA comprises a very GC-rich (75%) 5; region, while the 3; portion has a GC content of 50%. This gene encodes a protein with CPD photolyase activity when expressed in E. coli. The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5. One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues. The two RNAs occur in about equal amounts in O. sativa cells.

Positive association of CYP11B2 gene polymorphism with genetic predisposition to essential hypertension.

Predispositions to essential hypertension and cardiovascular diseases are possibly associated with gene polymorphisms of the renin-angiotensin system. Gene polymorphisms of angiotensinogen and angiotensin-converting enzyme genes have been suggested to be risk factors for hypertension and myocardial infarction. Concerning the polymorphism of aldosterone synthase (CYP11B2) gene, earlier studies have shown inconsistent results in terms of its relation to hypertension. In the present case-control study, we investigated the association of -344T/C polymorphism in the promoter region of human CYP11B2 gene with genetic predisposition to hypertension. The genotype of -344T/C polymorphism was determined in essential hypertension subjects (n=250) and normotensive subjects (n=221). The distributions of three genotypes (TT, TC, and CC) were significantly different between the hypertensive and the normotensive groups (chi(2)=9.61, P=0.008). Namely, the frequency of C allele was higher in the hypertensive patients than in the normotensive subjects (34.2 vs 26.5%, P=0.010). Our data suggest that the -344C allele of CYP11B2 gene polymorphism is associated with the genetic predisposition to develop essential hypertension.

Z and W chromosomes of chickens: studies on their gene functions in sex determination and sex differentiation.

Since the discovery of SRY/SRY as a testis-determining gene on the mammalian Y chromosome in 1990, extensive studies have been carried out on the immediate target of SRY/SRY and genes functioning in the course of testis development. Comparative studies in non-mammalian vertebrates including birds have failed to find a gene equivalent to SRY/SRY, whereas they have suggested that most of the downstream factors found in mammals including SOX9 are also involved in the process of gonadal differentiation. Although a gene whose function is to trigger the cascade of gene expression toward gonadal differentiation has not been identified yet on either W or Z chromosomes of birds, a few interesting genes have been found recently on the sex chromosomes of chickens and their possible roles in sex determination or sex differentiation are being investigated. It is the purpose of this review to summarize the present knowledge of these sex chromosome-linked genes in chickens and to give perspectives and point out questions concerning the mechanisms of avian sex determination.

Absence of Z-chromosome inactivation for five genes in male chickens.

In order to examine if Z-chromosome inactivation, which is analogous to X-chromosome inactivation in mammals, takes place in male birds having ZZ sex chromosomes, five Z-linked genes of chickens which are expressed in both sexes in certain tissues were selected: i.e. genes for growth hormone receptor, nicotinic acetylcholine receptor beta3, aldolase B, beta1,4-galactosyltransferase I, and iron-responsive element-binding protein (also known as cytosolic aconitase). Antisense or sense riboprobe was prepared from an intronic sequence of each gene and subjected to fluorescence in situ hybridization to nascent transcripts of each gene in a nucleus. Each antisense riboprobe hyridized to two spots of nascent RNA which corresponded to its gene loci on the two Z chromosomes in a majority of nuclei in a tissue of the male. The efficiency of detection of two spots per nucleus was comparable to that for the glyceraldehyde-3-phosphate dehydrogenase gene, an autosomal housekeeping gene. These results suggest strongly that Z-chromosome inactivation, i.e. virtual silence of transcription at one of the alleles, does not take place for these five Z-linked genes in male chickens.

Selective synthesis of 4-methoxyestrogen from 4-hydroxyestrogen.

The introduction of an oxygen atom into the C-6 position of 4-hydroxyestrogen allowed for the selective methylation of the two phenolic hydroxyl groups. When the 6-oxo derivative of 4-hydroxyestrone was benzylated in ethanol, only the 3-monobenzyl ether was obtained without formation of the 4-monobenzyl ether. Moreover, the 6-carbonyl group was further reduced to methylene almost quantitatively in the reaction of 4-acetoxy-6-oxoestrone 3-benzyl ether derivative with sodium borohydride. Therefore, 4-methoxyestrogen was synthesized by essentially combining these two reactions.

Molecular forms of plasma and urinary adrenomedullin in normal, essential hypertension and chronic renal failure.

OBJECTIVES: Human adrenomedullin precursor is converted to glycine-extended adrenomedullin (AM-Gly), an intermediate inactive form of adrenomedullin. Subsequently, AM-Gly is converted to active form of mature adrenomedullin (AM-m). The aim of the present study was to investigate (i) whether sex or age influences plasma and urinary AM-m and AM-Gly levels in normal subjects; (ii) the daytime variability of plasma AM-m and AM-Gly levels in normal subjects; (iii) AM-m and AM-Gly levels and its ratio in plasma and urine in normal subjects, individuals with essential hypertension (HT), and chronic renal failure (CRF); and (iv) the ratio of AM-m and AM-total (T) in plasma of various veins and aorta. METHODS: We measured plasma levels and urinary excretions of AM-m, AM-Gly and AM-T (AM-m + AM-Gly) by recently developed immunoradiometric assay in normal subjects (n = 81), HT (n = 28) and CRF (n = 30). We also determined the molecular forms of plasma adrenomedullin taken from various sites during angiography in patients with suspected renovascular hypertension (n = 9). RESULTS: There were no differences in plasma and urinary excretions of two molecular forms of adrenomedullin among sexes or ages in normal subjects. There was no daytime variation of plasma two molecular forms of adrenomedullin in normal subjects. Plasma AM-m, AM-Gly and AM-T levels were increased in patients with HT and CRF compared with normal subjects, whereas urinary AM-m, AM-Gly and AM-T excretions were decreased in patients with HT and CRF compared with normal subjects. Urinary AM-m: AM-T ratios were significantly higher than plasma AM-m: AM-T ratios. Plasma AM-m and AM-T levels taken from various veins were similar, and they were significantly higher than those of aorta, although there were no differences in plasma AM-Gly levels between aorta and veins. CONCLUSIONS: These results suggest that in normal subjects, and individuals with HT and CRF: (i) plasma and urinary excretions of AM-m and AM-Gly are not affected by age or sex; (ii) AM-m in parallel with AM-Gly is increased; (iii) urine contains a higher percentage of active adrenomedullin than plasma; and (iv) plasma AM-m may be partly metabolized in the lung.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus.

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Opposite effects of microtubule-stabilizing and microtubule-destabilizing drugs on biogenesis of mitochondria in mammalian cells.

Distribution of mitochondria as well as other intracellular organelles in mammalian cells is regulated by interphase microtubules. Here, we demonstrate a role of microtubules in the mitochondrial biogenesis using various microtubule-active drugs and human osteosarcoma cell line 143B cells and rat liver-derived RL-34 cells. Depolymerization of microtubules by nocodazole or colchicine, as well as 2-methoxyestradiol, a natural estrogen metabolite, arrested asynchronously cultured cells in G(2)/M phase of cell cycle and at the same time inhibited the mitochondrial mass increase and mtDNA replication. These drugs also inhibited the mitochondrial mass increase in the cells that were synchronized in cell cycle, which should occur during G(1) to G(2) phase progression in normal conditions. However, stabilization of microtubules by taxol did not affect the proliferation of mitochondria during the cell cycle, yet a prolonged incubation of cells with taxol induced an abnormal accumulation of mitochondria in cells arrested in G(2)/M phase of cell cycle. Taxol-induced accumulation of mitochondria was not only demonstrated by mitochondria-specific fluorescent dyes but also evidenced by the examination of cells transfected with yellow fluorescent protein fused with mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase (pEYFP) and by enhanced mtDNA replication. Two subpopulations of mitochondria were detected in taxol-treated cells: mitochondria with high Delta(psi)(m), detectable either by Mito Tracker Red CMXRos or by Green FM, and those with low Delta(psi)(m), detectable only by Green FM. However, taxol-induced increases in the mitochondrial mass and in the level of acetylated (alpha)-tubulin were abrogated by a co-treatment with taxol and nocodazole or taxol and colchicine. These data strongly suggest that interphase microtubules may be essential for the regulation of mitochondrial biogenesis in mammalian cells.

Effects of alpha-tocopherol on cisplatin-induced ototoxicity in guinea pigs.

Cisplatin (CDDP), an antitumor agent widely used in the treatment of head and neck cancers, has dose-limiting side effects such as ototoxicity and nephrotoxicity. Recently, evidence has been accumulated to demonstrate that these side effects are closely related to oxidative stress. In the present study, we attempted to suppress CDDP-induced ototoxicity and nephrotoxicity in guinea pigs by administering alpha-tocopherol, a naturally occurring antioxidant. Hartley albino guinea pigs (250 approximately 300 g) were treated with CDDP (4 mg/kg intraperitoneally (I.P.)) for 3 days in the presence and absence of alpha-tocopherol (50 mg/kg I.P.) injection for 6 days. The combined treatment of animals with alpha-tocopherol distinctly improved the CDDP-induced side effects. These were: loss of Preyer's reflex at high frequencies; distinct elevation of auditory brain stem response threshold at 16 kHz; increased lipid peroxidation in the cochlea determined by the malondialdehyde-thiobarbituric acid method; substantial losses of outer hair cells in the basal and second turns of the cochlea; fragmentation of nuclear DNA detected by the TUNEL method in cochlear hair cells and cells in the stria vascularis; and increases in serum BUN and Cr. These results strongly suggest that alpha-tocopherol suppresses CDDP-induced ototoxicity and nephrotoxicity via the suppression of the increased production of reactive oxygen species.

Analysis of pathological tremors using the autoregression model.

The usefulness of analysis of acceleration data using an autoregression model (AR) for differential diagnosis of Parkinson's disease and other diseases with tremors was investigated. The order of the AR model used in this study was 7, in accordance with Akaike's final prediction error criterion. The subjects included 19 patients with Parkinson's disease; 21 patients with essential tremor, which mainly appears in old people, as well as Parkinson's disease; and 13 healthy old people as a control group. The results of analysis of acceleration data showed that the first prediction coefficient, just as the main tremor frequency, was a useful parameter for differentiating patients in the Parkinson's disease patient group and essential tremor patient group. The seventh prediction coefficient was found to be a useful parameter for distinguishing pathological tremors observed in Parkinson's disease and essential tremor disease from physiological tremors observed in healthy people. Although the usefulness of other prediction coefficients for differential diagnosis of Parkinson's disease and other diseases with tremors has not yet been clarified, the results of this study showed that information obtained from AR model parameters in addition to information on main tremor frequency is useful for the diagnosis of Parkinson's disease.

Swelling of free-radical-induced megamitochondria causes apoptosis.

Recently, we have found that cultured cells from various sources exposed to free radicals become apoptotic in the presence of megamitochondria (MG). The purpose of the present study is to answer the following two questions: (1) Do functions obtained from the "MG fraction" isolated from normal mitochondria by a routine procedure represent the functions of MG since the fraction consists of enlarged and normal-size mitochondria? (2) What is the correlation between MG formation and apoptotic changes of the cell? In the present study the heavy fraction rich in mitochondria enlarged to varying degrees and the light fraction consisting mainly of normal-size mitochondria were isolated independently from the livers of rats treated with hydrazine for 4 days (4H animals) and 8 days (8H animals), and some functions related to apoptosis were compared. Results were as follows: (1) Mitochondria in both fractions obtained from 8H animals swelled far less in various media than those obtained from the controls, suggesting that the permeability transition pores had been opened before they were exposed to swelling media. (2) The membrane potential of mitochondria in both fractions obtained from 8H animals was distinctly decreased. (3) The rates of reactive oxygen species generation from mitochondria of both fractions in 4H animals were equally elevated, while those in 8H animals were equally decreased compared to those of controls. These results, together with morphological data obtained in the present study, suggest that enlarged and normal-size mitochondria are a part of MG and that the secondary swelling of MG causes the apoptotic changes in the cell.

Functional aspects of megamitochondria isolated from hydrazine- and ethanol-treated rat livers.

It is essential to analyze functions of megamitochondria (MG) to elucidate the mechanism of the formation of MG induced under various pathological conditions. The MG fraction obtained by a routine isolation procedure for normal mitochondria always consists of a mixed population of mitochondria enlarged to various degrees and also normal-sized ones. The purpose of the present study is to answer the question of whether or not data obtained from the MG fraction consisting of such a heterogeneous population of mitochondria with respect to their sizes really reflect functions of MG. In the present study mitochondria were obtained from the livers of rats treated with a 1% hydrazine diet for 8 days and those given 32% ethanol in drinking water for up to 2 months using various isolation procedures. Results obtained are summarized as follows: (i) mitochondria enlarged to various degrees and normal-sized ones are sometimes connected with each other by a narrow stalk in the hepatocyte of hydrazine-treated animals, and such connections are maintained to some extent when mitochondria are isolated; and (ii) mitochondria obtained from experimental animals by a routine isolation procedure for mitochondria ((700-7000)gR2"') and those obtained by alternative isolation procedure yielding the heavy ((500-2000)gR2"') and light ((2000-7000)gR2"') fractions show some functional similarities: decreases in the content of cytochrome a + a3; decreases in oxygen consumptions and phosphorylating abilities; decreases in monoamine oxidase and cytochrome c oxidase activities; lowered membrane potential of mitochondria; decreases in the rate of the generation of reactive oxygen species. These results may suggest that mitochondria enlarged to various degrees and normal-sized ones are functionally similar to each other and that the MG fraction obtained by a routine isolation procedure for normal mitochondria can be applied to the study of the function of MG.