Malaysians of Malay, Chinese, and Indian ancestries were electrophoretically phenotyped for Amy1 and saliva esterase region 1 (Set-1) from saliva, Amy2 from plasma, soluble and mitochondrial GOT and PGM3 from leukocyte and placenta. Kadazans and Bajaus, the indigenous people of Sabah, East Malaysia were surveyed for Amy2. Three types of variants were observed for Amy1, one type for Amy2. Only Indians were found to be polymorphic for Amy1. Two GOTs 2-1 and three GOTm 2-1 variants were found among 281 Chinese while three GOTm 2-1 variants were found among 311 Malays. Malaysian Malays, Chinese, and Indians were found to be polymorphic for Set-1 and PGM3. The gene frequencies in Malays are Set-1F=0.601 +/- 0.021, Set-1S = 0.399 +/- 0.021; PGM13 = 0.788 +/- 0.020, PGM23 = 0.212 +/- 0.020; in Chinese Set-1F = 0.497 +/- 0.028, Set-1S = 0.503 +/- 0.028; PGM13 = 0.745 +/- 0.24, PGM23 = 0.255 +/- 0.024; in Indians, Set-1F = 0.449 +/- 0.031, Set-1S = 0.551 +/- 0.031; PGM13 = 0.755 +/- 0.029, PGM23 = 0.245 +/- 0.029.
The ultrastructure of the pinealocytes of the Malaysian rat (Rattus sabanus), a mammal inhabiting a zone near the equator where the annual variations of daylength are inconspicuous, was examined and compared with that of pinealocytes of other mammals. On the basis of the presence of granular vesicles, only one population of pinealocytes was found. A large number of granular vesicles and vesicle-crowned rodlets is characteristic of the pinealocytes of this equatorial species. Vesicle-crowned rodlets are especially numerous in the endings of the pinealocyte processes and; they most often found in direct topographical connection with the perivascular spaces. The physiological significance of the presence of such large amounts of vesicle-crowned rodlets and of the secretory process characterized by the formation of granular vesicles is discussed.
A comparative study of four Malayan ferns, Christensenia aesculifolia (Bl.) Maxon, Tectaria singaporeana (Wall.) Ching, Abacopteris multilineata (Wall.) Ching and Hymenophyllum polyanthos Sw. from shady habitats and another four, Dicranopteris linearis (Burm.) Und., Lygodium scandens (L.) Sw., Blechnum orientate Linn, and Stenochlaena palustris (Burm.) Bedd. from sunlit habitats showed that the total chlorophyll content expressed on a gram fresh weight basis was greater in the shade ferns. There was little difference in the chlorophyll content between the sun and shade ferns when it was expressed on a per unit leaf area basis. The protein and protohaem content was greater in the sun ferns. Measurements of the in vitro photochemical activities of the photosystems I and II in isolated chloroplasts by means of an oxygen electrode showed higher rates in the sun ferns. As determined by spectrophotometric analysis, the photosynthetic cytochrome content from isolated chloroplasts was greater in the sun ferns. The results indicate that the sun ferns have physiological characteristics favouring greater capacity for photosynthesis. Mitochondria isolated from the sun ferns showed faster rates of electron transport using exogenous NADH as substrate.
Margosa oil (MO), a fatty acid-rich extract of the seeds of the neem tree and a reported cause of Reye's syndrome, has been used in the induction of an experimental model of Reye's syndrome in rats. It has been reported that MO causes a decrease in in vivo mitochondrial enzyme activity similar to that seen in Reye's syndrome. We have attempted to uncover some of the biochemical mechanisms of MO's toxicity by examining its effect in vitro on isolated rat liver mitochondria. Male rat liver mitochondria were isolated by centrifugation; oxygen uptake, reduced forms of cytochrome b, c + c1, a + a3, and flavoprotein, intramitochondrial concentrations of acetyl coA, acid-soluble coA, acid-insoluble coA, and ATP content were measured after incubation with and without MO. Our results reveal that MO is a mitochondrial uncoupler. State 4 respiration was increased while the respiratory control ratio was decreased. The intramitochondrial content of ATP was also decreased. There were substantial changes in the reduction of the respiratory chain components after incubation of mitochondria with MO. This decelerative effect on mitochondrial electron transport was alleviated by the addition of coenzyme Q and/or carnitine. These effects of MO on mitochondrial respiration may be due to changes in fatty acid metabolism caused by MO as MO caused a shift in the proportion of acid-soluble or acid-insoluble coA esters. Supplementary therapy with L-carnitine and coenzyme Q may be useful in the management of MO-induced Reye's syndrome.
Cyclic AMP phosphodiesterase (PDE) activity was assayed in the plasma membrane, mitochondrial and microsomal fractions of rat brain. The specific activity of the enzyme was highest in the plasma membrane fraction followed by mitochondrial and then the microsomal fraction. Phosphodiesterase activity of all three fractions was reduced after pretreatment with lecithinase C (PCase) from Clostridium perfringens but less markedly affected by the pretreatment with sphingomyelinase (SMase) from human placenta. The PDE activity of the plasma membrane fraction was more sensitive to PCase treatment compared with the other two particulate fractions, which showed only a slight loss of activity. Temperature seemed to affect PDE activity of the plasma membrane. The enzyme was quite stable at 30 degrees C but its activity dropped by approximately 46% at 37 degrees C after 90 min of incubation. Pretreatment of the plasma membrane at 30 degrees C with PCase at a concentration of more than 5 U caused a marked loss of PDE activity and the decrease in activity reached a plateau at concentrations above 10 U.
The effect of palm oil, a widely used vegetable oil, rich in tocotrienols, on peroxidation potential of rat liver was examined. Long-term feeding of rats with palm oil as one of the dietary components significantly reduced the peroxidation potential of hepatic mitochondria and microsomes. As compared to hepatic mitochondria isolated from rats fed control or corn oil-rich diet, those from palm oil-fed group showed significantly less susceptibility to peroxidation induced by ascorbate and NADPH. However, in microsomes, only NADPH-induced lipid peroxidation was significantly reduced in rats fed palm oil rich-diet. Though the accumulation of thiobarbituric acid reactive substances during ascorbate-induced lipid peroxidation in mitochondria from rats fed corn oil-rich diet supplemented with tocotrienol-rich fraction (TRF) of palm oil was similar to that of control rats, the initial rate of peroxidation was much slower than those from control or corn oil fed diets. Our in vitro studies as well as analyses of co-factors related to peroxidation potential indicated that the observed decrease in palm oil-fed rats may be due to increased amount of antioxidants in terms of tocotrienol as well as decrease in the availability of substrates for peroxidation.
The structure and distribution of organelles within developing goat oocytes at various stages of incubation were studied. In oocytes with 5 or more layers of cumulus cells, at 0 h of incubation, the zona pellucida had developed although zonation was not evident. Lipid bodies were present but no mitochondria were observed. At 20 h, the zona pellucida had differentiated into thicker and thinner regions. Clusters of membrane-bound electron-transparent bodies were present in the perivitelline space. The mitochondria were fully developed, distributed evenly and usually in close proximity with dilated endoplasmic reticula. Cortical granules were distributed at the periphery. At 40 h of incubation, a number of mitochondria was hooded. In oocytes of 2 to 4 layers of cumulus cells at 0 h, the zona pellucida was penetrated by cumulus cell processes, and the mitochondria were not well developed. However, in 20-h incubated oocytes, fully developed mitochondria, many of which were hooded, could be observed. Clusters of membrane-bound electron-transparent bodies were also observed, while cortical granules were at the periphery. In cumulus-free oocytes, zonation within the zona pellucida was indistinct. Very few vesicles and lipid bodies were observed. At 20 h, mitochondria were sparsely distributed and were not well developed and lacked cristae. At 40 h, the zona pellucida was less compact, and the membrane-bound electron-transparent bodies were less numerous compared with those of the other groups. Endoplasmic reticula were not dilated, and cortical granules were few and had no definite pattern of distribution.
Heart mitochondria freshly isolated from ginseng treated rats respired higher at ADP-induced, state 3 respiratory rates and with greater respiratory indices. These mitochondria were less susceptible to experimentally-induced functional impairment. Control heart mitochondria incubated with ginseng extract also showed that ginseng prevented mitochondria from incubation induced deterioration with NAD-linked substrates. Comparison of force of contraction of isolated, perfused and electrically paced hearts showed that deterioration of the force of heart contraction was consistently smaller throughout the experiment in hearts from ginseng treated rats. These results indicated that Panax ginseng was able to delay experimentally induced heart mitochondrial impairment and muscle contraction deterioration.
A study of 101 sera from 69 Malay, 14 Chinese and 18 Indian healthy adult Malaysians was undertaken to determine the frequency of antinuclear antibodies (ANA), antimitochondrial antibodies (AMA), antismooth muscle antibodies (SMA) and antiparietal cell antibodies (APCA). There were 67 females and 34 males with a mean age of 31.7 years (+/-8.6). ANA was assayed by immunofluorescence (IF) using both mouse liver and HEp-2 cell substrates. AMA, SMA and APCA were also tested by IF using composite sections from mouse liver, kidney and stomach substrates. Analysis showed 6.9% were positive for ANA at a titre of 1:40 with HEp-2 while only 1.9% were detected using mouse liver. 9.9% had detectable AMA from titres 1:10 to 1:90. None of them had detectable SMA and only 1 (0.09%) had APCA at a titre of 1:80. This study suggests that a diagnosis of an autoimmune disorder has to be cautiously made taking into consideration that autoantibodies are present in low titres in the healthy population.
This report describes a 56-yr-old man with a dominantly inherited disorder affecting four generations and characterized by bilateral ptosis and dysphagia. Muscle biopsy showed only minor light microscopic abnormalities but electron microscopy revealed fibres containing paracrystalline mitochondrial inclusions. Southern analysis of mitochondrial DNA obtained from muscle did not reveal mitochondrial gene deletions. An extensive search eventually identified the characteristic intranuclear filaments of oculopharyngeal muscular dystrophy (OPMD). Abnormal mitochondria are non-specific epiphenomena in OPMD but a potential source of confusion with a late-onset mitochondrial cytopathy. This case further emphasizes the necessity for a diligent search for the diagnostic intranuclear filaments when oculopharyngeal muscular dystrophy is suspected clinically.
The intracellular movements of pregnenolone in rat testes were investigated. Whole testes were incubated in the presence or absence of pregnenolone (2.5mM) in the medium for 120 min (in some studies 30, 60, and 90 min). The testes were homogenised, subcellular fractions prepared and analysed in quadruplicate for steroid content by gas chromatography-mass spectrometry with selected ion monitoring. Quantification of pregnenolone and 11 of its metabolites, obtained from non-incubated whole testes, provided values for endogenous amounts. Pregnenolone was the only steroid of quantitative importance found initially in the mitochondrial fraction but was subsequently found in the microsomal fraction, where metabolism occurred. Identification and quantification of metabolites indicated that both classical pathways for testosterone production were operating, with the 4-en-3-oxosteroid pathway predominating. By 120 min, virtually all pregnenolone metabolites, including pregnenolone itself, were found in the cytosol, consistent with an overall movement from mitochondria to endoplasmic reticulum to cytosol.
Styryl-lactones such as goniothalamin represent a new class of compounds with potential anti-cancer properties. In this study, we investigated the mechanisms of goniothalamin (GTN), a plant styryl-lactone induced apoptosis in human promyelocytic leukemia HL-60 cells. This plant extract resulted in apoptosis in HL-60 cells as assessed by the externalisation of phosphatidylserine. Using the mitochondrial membrane dye (DIOC(6)) in conjunction with flow cytometry, we found that GTN treated HL-60 cells demonstrated a loss of mitochondrial transmembrane potential (Deltapsi(m)). Further immunoblotting on these cells showed activation of initiator caspase-9 and the executioner caspases-3 and -7. Pretreatment with the pharmacological caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) abrogated apoptosis as assessed by all of the apoptotic features in this study. In summary, our results demonstrate that goniothalamin-induced apoptosis occurs via the mitochondrial pathway in a caspase dependent manner.
For many liver malignancies, major hepatectomy is the usual therapy. Although a normal liver has a tremendous capacity for regeneration, liver hepatectomy in humans is usually carried out on a diseased liver and, in such cases, liver regeneration takes place in a cirrhotic remnant. Mitochondrial function in cirrhotic livers shows a variety of changes compared to control livers. This study investigated how mitochondrial respiratory function and antioxidant capacity change following partial hepatectomy of cirrhotic livers, because liver regeneration requires greater energy demands and control of oxidative stress. Cirrhosis was induced in male Wistar-Furth rats by administration of thioacetamide. NADH-cytochrome c reductase activity, mitochondrial glutathione peroxidase activity and mitochondrial GSH levels were all significantly lowered in cirrhotic livers and in the cirrhotic remnants up to 72 h after 70% hepatectomy when compared to the corresponding controls. Lower respiratory control ratios with succinate as substrate were also observed from 6 to 48 h post-hepatectomy. At 24 h post-hepatectomy, higher levels of lipid peroxidation were observed. We conclude that, compared to the controls, cirrhotic livers have diminished oxidative phosphorylation capabilities due to changes in NADH and FADH(2)-linked respiration as well as impaired antioxidant defenses following partial hepatectomy. Both of these factors, if critical, could then impede liver regeneration.
A recent dispersal of modern humans out of Africa is now widely accepted, but the routes taken across Eurasia are still disputed. We show that mitochondrial DNA variation in isolated "relict" populations in southeast Asia supports the view that there was only a single dispersal from Africa, most likely via a southern coastal route, through India and onward into southeast Asia and Australasia. There was an early offshoot, leading ultimately to the settlement of the Near East and Europe, but the main dispersal from India to Australia approximately 65,000 years ago was rapid, most likely taking only a few thousand years.
The pineal product melatonin has remarkable antioxidant properties. It scavenges hydroxyl, carbonate and various organic radicals, peroxynitrite and other reactive nitrogen species. Melatonyl radicals formed by scavenging combine with and, thereby, detoxify superoxide anions in processes terminating the radical reaction chains. Melatonin also enhances the antioxidant potential of the cell by stimulating the synthesis of antioxidant enzymes like superoxide dismutase, glutathione peroxidase and glutathione reductase, and by augmenting glutathione levels. The decline in melatonin production in aged individuals has been suggested as one of the primary contributing factors for the development of age-associated neurodegenerative diseases, e.g., Alzheimer's disease. Melatonin has been shown to be effective in arresting neurodegenerative phenomena seen in experimental models of Alzheimer's disease, Parkinsonism and ischemic stroke. Melatonin preserves mitochondrial homeostasis, reduces free radical generation, e.g., by enhancing mitochondrial glutathione levels, and safeguards proton potential and ATP synthesis by stimulating complex I and IV activities. Therapeutic trials with melatonin have been effective in slowing the progression of Alzheimer's disease but not of Parkinson's disease. Melatonin's efficacy in combating free radical damage in the brain suggests that it may be a valuable therapeutic agent in the treatment of cerebral edema after traumatic brain injury.
Increased oxidative stress and mitochondrial dysfunction have been identified as common pathophysiological phenomena associated with neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). As the age-related decline in the production of melatonin may contribute to increased levels of oxidative stress in the elderly, the role of this neuroprotective agent is attracting increasing attention. Melatonin has multiple actions as a regulator of antioxidant and prooxidant enzymes, radical scavenger and antagonist of mitochondrial radical formation. The ability of melatonin and its kynuramine metabolites to interact directly with the electron transport chain by increasing the electron flow and reducing electron leakage are unique features by which melatonin is able to increase the survival of neurons under enhanced oxidative stress. Moreover, antifibrillogenic actions have been demonstrated in vitro, also in the presence of profibrillogenic apoE4 or apoE3, and in vivo, in a transgenic mouse model. Amyloid-beta toxicity is antagonized by melatonin and one of its kynuramine metabolites. Cytoskeletal disorganization and protein hyperphosphorylation, as induced in several cell-line models, have been attenuated by melatonin, effects comprising stress kinase downregulation and extending to neurotrophin expression. Various experimental models of AD, PD and HD indicate the usefulness of melatonin in antagonizing disease progression and/or mitigating some of the symptoms. Melatonin secretion has been found to be altered in AD and PD. Attempts to compensate for age- and disease-dependent melatonin deficiency have shown that administration of this compound can improve sleep efficiency in AD and PD and, to some extent, cognitive function in AD patients. Exogenous melatonin has also been reported to alleviate behavioral symptoms such as sundowning. Taken together, these findings suggest that melatonin, its analogues and kynuric metabolites may have potential value in prevention and treatment of AD and other neurodegenerative disorders.
The authors describe the case of a 50-year-old man with chronic progressive external ophthalmoplegia (CPEO), diabetes mellitus (DM), and coronary artery disease. The patient had no cardiac conduction abnormalities. During coronary artery bypass surgery, his heart and two skeletal muscles were biopsied. All three muscles showed ragged red fibers. The heart muscle showed significant glycogen accumulation. Analysis of mitochondrial DNA (mtDNA) showed a 5019-base-pair deletion, with no duplications. There were morphologically abnormal mitochondria in all 3 muscles, with clinically apparent difference in preservation of function. The combination of diabetes mellitus and mtDNA deletion is fortuitous, as they can be causally linked. The cardiac pathology allows speculation about the possible adaptive processes that may occur in the heart in DM. There are few reported cases with CPEO and excess glycogen in the heart. Most show deposition of fat and poorer clinical outcomes as compared to those with glycogen deposition. This observation may lend support to the hypothesis that in the myocardium, adaptive responses are mediated via changes in glucose handling, whereas alterations in fat metabolism likely represent maladaptation.
Liver involvement, a common feature in childhood mitochondrial hepatopathies, particularly in the neonatal period, may manifest as neonatal acute liver failure, hepatic steatohepatitis, cholestasis, or cirrhosis with chronic liver failure of insidious onset. There are usually significant neuromuscular symptoms, multisystem involvement, and lactic acidemia. The liver disease is usually progressive and eventually fatal. Current medical therapy of mitochondrial hepatopathies is largely ineffective, and the prognosis is usually poor. The role of liver transplantation in patients with liver failure remains poorly defined because of the systemic nature of the disease that does not respond to transplantation. Several specific molecular defects (mutations in nuclear genes such as SCO1, BCS1L, POLG, DGUOK, and MPV17 and deletion or rearrangement of mitochondrial DNA) have been identified in recent years. Prospective, longitudinal multicenter studies will be needed to address the gaps in our knowledge in these rare liver diseases.
This study was conducted to identify and to compare the mitochondrial morphological alterations in livers of rats treated with various doses of diclofenac and ibuprofen. Hundred and forty-four male Sprague Dawley rats were dosed with 3, 5 and 10 mg kg(-1) diclofenac and ibuprofen in saline via intraperitoneal injection for 15 days. The control group was administered with saline in a similar manner. Four rats were euthanised every 3 days until day 15. While 200 mg kg(-1) diclofenac and ibuprofen-treated rats (n = 4) were euthanized 10 h posttreatment. The livers were removed, cleaned and a section across the right lobe was taken and fixed in 4% (v/v) glutaraldehyde for electron microscopy analysis and the remaining samples were kept at -80 degrees C for Western blot analysis. Five milligram per kilogram and 10 mg kg(-1) diclofenac-administered rats for 15 days revealed the presence of enlarged mitochondria, irregular and ruptured mitochondrial membranes. While rats administered with 10 mg kg(-1) ibuprofen also showed the presence of mitochondria with irregular membrane structure and ruptured membranes. Western blotting analysis of mitochondrial fractions revealed the expression of cytochrome c in all samples and complete absence of cytochrome c expression in the cytosolic fraction of all samples after day 15. Analysis in 200 mg kg(-1) diclofenac and ibuprofen-treated groups, revealed expression of cytochrome c in both mitochondrial and cytosolic fractions. This observation indicates that both diclofenac and ibuprofen may alter the morphology of mitochondria, leading to cytochrome c release into the cytosol. Further studies needs to be conducted to investigate on the activity of the mitochondria following both treatments.
We report the biochemical profiling in two siblings with mitochondrial 2-methylacetoacetyl-CoA thiolase deficiency. Organic aciduria typical of this rare inborn error metabolism was found when the elder sibling presented with an episode of severe ketoacidosis at 20 months of age, which consisted of excessive excretion of ketones, tiglylglycine, 2-methyl-3-hydroxybutyrate, and 2-methylacetoacetate. Blood acylcarnitiness profile showed elevation of C5OH-carnitine, which represents 2-methyl-3-hydroxybutyrylcarnitine. A similar biochemical profile was identified in the younger sibling during screening although he had only mild clinical symptoms. Both patients reported a favourable outcome on follow-up.