Infantile familial cardiomyopathy due to mitochondrial complex I and IV associated deficiency

Two brothers, aged 27 and 20 months, born from consanguineous healthy parents, presented with cardiomyopathy, lactic acidosis and carnitine abnormalities in serum and muscle, without clinical evidence of muscle involvement. The histochemical reaction for cytochrome c oxidase (COX) activity was negative in all muscle fibres, although the holoenzyme and subunits were present at a normal level, as shown by immunocytochemistry. The COX activity was, respectively, 5 and 25% of control values, in muscle biopsies. Partial deficiency of complex IV was confirmed in fresh isolated muscle mitochondria from patient 2 and was associated with a defect of complex I. Patient 1 died at age 3 yr 6 months. Partial improvement of cardiomyopathy in patient 2 was obtained under carnitine therapy, but seizures occurred and CT scan and magnetic resonance imaging (MRI) revealed thalamic hypodensity. Thus, the disorder appears to be progressive despite the clinical stabilization of the cardiomyopathy. This further demonstrates the complexity and clinical heterogeneity of combined respiratory chain complex deficiencies.     

Nature of inhibition of mitochondrial respiratory complex I by 6-Hydroxydopamine

The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron (III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.     

Therapy of complex I deficiency: peripheral neuropathy during dichloroacetate therapy

A therapeutic trial with polyvitamins and dichloroacetate (DCA) in combination with thiamine in a 13-year-old girl with complex I deficiency is reported. The polyvitamin therapy included thiamine, riboflavin, ascorbate, coenzyme Q 10 and carnitine. This therapeutic regine was used over a period of 17 months without any effect. Although DCA lowered the lactate concentration in blood and CNS--measured by magnetic resonance spectroscopy--no clinical benefit was achieved. After 20 weeks of DCA therapy a distal polyneuropathy with areflexia developed although 100 mg thiamine daily as comedication was given from the beginning of DCA therapy. Nerve conduction velocity of the peroneal nerve was not detectable, sensible evoked potentials of the tibialis posterious nerve were normal. This side-effect resolved completely within 6 months after omission of DCA. Our observation suggests a direct toxic effect of DCA only on the peripheral nervous system in our patient since several cerebral MRI and magnetic resonance spectroscopy studies showed no abnormalities. CONCLUSION. DCA lowers the lactate concentration in children with complex I deficiency of the respiratory chain in a dose of 100 mg/kg body weight without clinical benefit. Reversible peripheral polyneuropathy may develop under DCA therapy despite thiamine medication.     

Anesthesia for a child with complex I respiratory chain enzyme deficiency

Computed tomography (CT) excretory urography was performed in five adult female dogs after intravenous injection of a bolus of four different doses of water-soluble iodinated contrast medium (100, 200, 400, and 800 mgI/kg). CT images centered over the urinary bladder were performed before injection and 1, 3, 5, 7, 9, 11, 15, 20, 25, 30, 40, 50, and 60 minutes after injection. Opacification of both ureters was evaluated by measuring maximum CT number of individual ureters at each time. Time opacification curves were generated for each dose. Best opacification of the ureters was obtained with 400 and 800 mgI/kg, with a constant peak at 3 minutes and durable opacification for 1 hour. Insufficient opacification was obtained with lower dose of 100 and 200 mgI/kg.     

Thiol oxidation and loss of mitochondrial complex I precede excitatory amino acid-mediated neurodegeneration

Human ingestion of "chickling peas" from the plant Lathyrus sativus, which contains an excitatory amino acid, L-BOAA (L-beta-N-oxalylamino-L-alanine), leads to a progressive corticospinal neurodegenerative disorder, neurolathyrism. Exposure to L-BOAA, but not its optical enantiomer D-BOAA, causes mitochondrial dysfunction as evidenced by loss of complex I activity in vitro in male mouse brain slices and in vivo in selected regions of mouse CNS (lumbosacral cord and motor cortex). Loss of complex I activity in lumbosacral cord after L-BOAA administration to mice was accompanied by concurrent loss of glutathione. The inhibited complex I activity in mitochondria isolated from lumbosacral cord of animals treated with L-BOAA rebounded after incubation with the thiol-reducing agent dithiothreitol, indicating that oxidation of protein thiols to disulfides was responsible for enzyme inhibition. The inhibition of complex I could be abolished by pretreatment with antioxidant thiols such as glutathione ester and alpha-lipoic acid. Chronic treatment of male mice, but not female mice, with L-BOAA resulted in loss of complex I activity and vacuolation and dendritic swelling of neurons in the motor cortex and lumbar cord, paralleling the regionality of the aforementioned biochemical effects on CNS mitochondria. These results support the view that thiol oxidation and concomitant mitochondrial dysfunction (also implicated in other neurodegenerative disorders), occurring downstream of glutamate receptor activation by L-BOAA, are primary events leading to neurodegeneration. Maintenance of protein thiol homeostasis by thiol delivery agents could potentially offer protection against excitotoxic insults such as those seen with L-BOAA.     

Characterization of the ubiquinone reduction site of mitochondrial complex I using bulky synthetic ubiquinones

A wide variety of alkyl derivatives of Q2 (6-geranyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone) and DB (6-n-decyl-2, 3-dimethoxy-5-methyl-1,4-benzoquinone), in which methoxy groups of the 2- and/or 3-positions of the quinone ring were replaced by other bulky alkoxy groups from ethoxy to butoxy, were prepared by novel synthetic procedures. Electron-accepting activities of the bulky quinones were investigated with bovine heart mitochondrial complex I and its counterpart of Paracoccus denitrificans(NDH-1) to elucidate structural and functional features of the quinone reduction site of the enzymes. The bulky quinone analogues served as sufficient electron acceptors from the physiological quinone reduction site of bovine complex I. Considering the very poor activities of even the ethoxy derivatives as substrates for other respiratory enzymes such as mitochondrial complexes II and III [He, D. Y., Gu, L. Q., Yu, L., and Yu, C. A. (1994) Biochemistry 33, 880-884], this result indicated that the quinone reduction site of bovine complex I is spacious enough to accommodate bulky exogenous substrates. In contrast to bovine complex I, bulky quinone analogues served as poor electron acceptors with Paracoccus NDH-1. These observations indicated that bovine complex I recognizes the substrate structure with poor specificity. The substituent effects in the 2- and 3-positions of the quinone ring on the electron-transfer activity with bovine complex I differed significantly between Q2 and DB series despite having the same total number of carbon atoms in the side chain. The inhibitory effect involving Q2 due to its geranyl side chain was markedly diminished by structural modifications of the quinone ring moiety. These findings indicate that the side chain plays a specific role in the redox reaction and that the quinone ring and side-chain moieties contribute interdependently to binding interaction. Moreover, structural dependency of the proton-pumping activity of the quinone analogues was comparable to that of the electron-transfer activity with bovine complex I, indicating that the mechanism of redox-driven proton-pumping does not differ depending upon the substrate structure.     

Amylose-iodine complex. I. Sedimentation behavior

DNA-synthesizing cells in the gonads of the ascidian Styela clava were labeled with tritiated thymidine and detected with autoradiography. In the testis, spermatogonia and primary spermatocytes are labeled after 1 hr. Labeled spermatozoa occur in the lumen of the testis follicles after 10 days and in the sperm ducts after 20 days. In the ovary, only germ cells (oogonia and pre-leptotene primary oocytes) and follicle cells are labeled after 1 hr. By 60 days, oocytes with basophilic cytoplasm (15-65 mu in diameter) are labeled; test cells embedded in larger eosinophilic oocytes (150 mu in diameter) are also labeled. Germ cells give rise to both oocytes and follicle cells. Through continued cell division, follicle cells give rise to test cells.     

Cellular and dendritic growth: Part I. Experiment

An Al-4.1 mass pct Cu alloy was unidirectionally solidified under various growth rates. Features, such as tip radius of curvature, primary arm spacing, and tip concentration were measured as functions of growth rate. Dependence of tip radius on growth rate was different between cells and dendrites. Measured tip radius and primary arm spacing were maximum at the cellular-dendritic transition. Tip concentration, however, monotonously decreases with growth rate. Linear relationships between tip radius and characteristic dimensions of dendrites like core diameter, half length of tip arc, and the first secondary arm spacing are obtained to determine what affects growth rate, convection, and gravity segregation. Experimental results are compared with current theoretical models for dendrite growth under controlled solidification. It was determined that the measured tip radius is larger than that of theoretical predictions at fast growth rate, but the measured tip concentration is in good agreement with theoretical predictions.     

Human beta-glucuronidase. I. Recognition and uptake by animal fibroblasts suggests animal models for enzyme replacement studies

As part of an effort to develop an animal model for studies of uptake of human beta-glucuronidase, fibroblasts were established from primary explants of connective tissue from nine different animal species, and examined for their ability to take up human platelet beta-glucuronidase. Endogenous fibroblast beta-glucuronidase was inactivated by heating extracts to 65 degrees for 30 min. Human beta-glucuronidase was stable to this treatment. Uptake of human beta-glucuronidase by animal fibroblasts was measured as heat-stable beta-glucuronidase present in fibroblasts after exposure to partially purified human platelet beta-glucuronidase for 48 hr. Althought all animal fibroblasts examined exhibited some uptake capacity for human beta-glucuronidase, the uptake capacity of different animal fibroblasts varied over a 10-fold range. The uptake capacity of bovine fibroblasts was at least 80% that of human fibroblasts. Rat and hamster fibroblasts showed about half the uptake capacity of human fibroblasts. The rat fibroblasts resembled the human fibroblasts in the kinetics of uptake of hihg uptake (platelet) enzyme, poor uptake of human placental enzyme, and lack of appreciable turnover of enzyme taken up over 4 days. Heating extracts of rat organs containing added human beta-glucuronidase at 65 degrees selectively inactivated rat enzyme.     

Inhibition of mitochondrial complex I may account for IDDM induced by intoxication with the rodenticide Vacor

Human intoxication with the rodenticide Vacor [N-3-pyridylmethyl-N'-p-nitrophenyl urea or 1-(4-nitrophenyl)-3-(3-pyridylmethyl) urea] induces acute IDDM. We report here that Vacor specifically inhibits the NADH:ubiquinone reductase activity of complex I in mammalian mitochondria. The activity of other respiratory enzymes of mitochondria is unaffected by Vacor at concentrations that completely inhibit the redox and energetic function of complex I. Vacor inhibition of complex I activity quantitatively correlates with the inhibition of insulin release in insulinoma cells and pancreatic islets and is also consistent with the doses reported in cases of human poisoning. These results indicate that the toxic and diabetogenic action of Vacor primarily derives from the inhibition of mitochondrial respiration of NAD-linked substrates in the high-energy demanding cells of the pancreatic islets. This newly identified mechanism of the pathological effects resulting from Vacor intoxication could constitute a paradigm in which to understand environmental or metabolic causes of IDDM.     

Human C-peptide. Part I: Radioimmunoassay

Synthetic human C-peptide bearing a Tyrosine group at its amino end is labelled with 125iodine using chloramin T or hydrogen peroxide and lactoperoxidase. The results are compared applying both methods. Antiserum to synthetic human C-peptide (without Tyrosine) which was partially compared to rabbit albumin, is raised in guinea pigs and goats. Goats show to be superior to guinea pigs concerning antibody production. The so-called "hook effect" phenomenon is observed in setting up the standard curves for the radioimmunoassay. Monotonically decreasing standard curves are obtained on dilution of antiserum with a high antibody titer which was produced by repeated immunization in goats. Free C-peptide and C-peptide bound to antiserum are separated with the anxion exchange resin Amberlite. Using this separation technique we excluded unspecific binding of labelled C-peptide to protein fractions in serum of diabetics. The sensitivity of our radioimmunoassay is approx. 0.3 ng C-peptide/ml serum. Intra- and interassay variability are below 10%. Human proinsulin is the only substance found to crossreact with the antiserum.     

Nuclear genes of human complex I of the mitochondrial electron transport chain: state of the art

Exposure to aromatic amines is considered a major risk factor for the development of bladder cancer. In this study, we have analysed the pattern of point mutations in several tumour genes in 21 cases of bladder cancer arising among western European workers exposed to aromatic amines in an attempt to determine whether this exposure may be associated with a unique spectrum of mutations. Of the four genes analysed (p53, p16MTS1, p21WAF1 and H-ras), only p53 showed a high frequency of mutations (in 8 out of 21 cases, 38%). Two mutations were found in p16, one in H-ras and none in p21 exon 3. All mutations were at G:C base pairs, mostly at non-CpG residues. This spectrum of mutations, which is highly suggestive of an involvement of exogenous carcinogens, is however identical to the spectrum of p53 mutations detected in bladder cancers of the general population. In exposed workers, p53 mutations were associated with tumour grade and with high occupational and tobacco exposure. Taken together, our data suggest that the same carcinogens may be responsible for the development of bladder cancers in workers exposed to aromatic amines and in the general population.     

Human pancreatic alpha-amylase. I. Purification and characterization

alpha-Amylase was extracted from human pancreas and purified by using ammonium sulfate fractionation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography. The enzyme was shown to be homogenous by three different criteria: polyacrylamide disc gel electrophoresis, SDS polyacrylamide gel electrophoresis and analytical ultracentrifugation. The values of SO20,w, D20,w, v, and frictional ration of the enzyme were calculated to be 5.01S, 7.56D, 0.718 ml g-1 and 1.10, respectively. The molecular weight of the alpha-amylase was determined by three different methods: sedimentation velocity-diffusion, conventional sedimentation equilibrium and SDS polyacrylamide gel electrophoresis and was found to be 57,850; 50,100 and 53,200 g mole-1, respectively (average value 53,700). The amino acid composition of the enzyme was determined and compared with those of alpha-amylases from various other sources.     

Numerical solution of structured population models. I. Age structure

Numerical methods are presented for a general age-structured population model with demographic rates depending on age and the total population size. The accuracy of these methods is established by solving problems for which alternate solution techniques are available and are used for comparison. The methods reliably solve test problems with a variety of dynamic behavior. Simulations of a blowfly population exhibit cyclic fluctuations, whereas a simulated squirrel population reaches a stable age distribution and stable equilibrium population size. Life-history attributes are easily studied from the computed solutions, and are discussed for these examples. Recovery of a stressed population back to equilibrium is examined by computing the transition in age structure, and the transient behavior of other properties of the population such as the per capita growth rate, the average age, and the generation length.     

10-Oximeguanacone, the first nitrogenated acetogenin derivative found to be a potent inhibitor of mitochondrial complex I

BACKGROUND: Several retrospective studies have claimed that extracorporeal photopheresis (ECP) prolongs survival in patients with erythrodermic cutaneous T-cell lymphoma. In a retrospective study of 44 patients with Sézary syndrome, we compared survival in patients treated with ECP with that of patients treated conventionally at the same institute. All patients had genotypic evidence of a peripheral blood T-cell clone. OBSERVATIONS: Twenty-nine patients received ECP (group 1); 15 patients did not receive ECP, 8 patients when ECP was available (group 2) and 7 before ECP was available (group 3). Forty-three of 44 patients received other conventional treatments. Median survival from diagnosis of Sézary syndrome was 39 months in group 1, 22 months in group 2, and 27.5 months in group 3 (Kaplan-Meier analysis). Cox regression analysis showed no significant difference between the 3 groups after correcting for age, sex, and initial Sézary cell count (hazard ratio, 0.56; 95% confidence interval, 0.26-1.17; P = .12). CONCLUSIONS: This study does not support the contention that ECP prolongs survival in patients with Sézary syndrome. The median survival in the ECP-treated group is considerably less than that reported in other published series, possibly because genotypic evidence of clonality in the peripheral blood was required for inclusion in this study. We believe that a randomized trial comparing ECP with standard chemotherapy is urgently needed.