Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants.

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III2/IV in yeast or I/III2/IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes.

Regulation of the aldehyde dehydrogenase gene (aldA) and its role in the control of the coinducer level necessary for induction of the ethanol utilization pathway in Aspergillus nidulans.

Expression of the structural genes for alcohol and aldehyde dehydrogenase, alcA and aldA, respectively, enables the fungus Aspergillus nidulans to grow on ethanol. The pathway-specific transcriptional activator AlcR mediates the induction of ethanol catabolism in the presence of a coinducing compound. Ethanol catabolism is further subject to negative control mediated by the general carbon catabolite repressor CreA. Here we show that, in contrast to alcA and alcR, the aldA gene is not directly subject to CreA repression. A single cis-acting element mediates AlcR activation of aldA. Furthermore, we show that the induction of the alc gene system is linked to in situ aldehyde dehydrogenase activity. In aldA loss-of-function mutants, the alc genes are induced under normally noninducing conditions. This pseudo-constitutive expression correlates with the nature of the mutations, suggesting that this feature is caused by an intracellular accumulation of a coinducing compound. Conversely, constitutive overexpression of aldA results in suppression of induction in the presence of ethanol. This shows unambiguously that acetaldehyde is the sole physiological inducer of ethanol catabolism. We hypothesize that the intracellular acetaldehyde concentration is the critical factor governing the induction of the alc gene system.

The CreA repressor is the sole DNA-binding protein responsible for carbon catabolite repression of the alcA gene in Aspergillus nidulans via its binding to a couple of specific sites.

Carbon catabolite repression is mediated in Aspergillus nidulans by the negative acting protein CreA. The CreA repressor plays a major role in the control of the expression of the alc regulon, encoding proteins required for the ethanol utilization pathway. It represses directly, at the transcriptional level, the specific transacting gene alcR, the two structural genes alcA and aldA, and other alc genes in all physiological growth conditions. Among the seven putative CreA sites identified in the alcA promoter region, we have determined the CreA functional targets in AlcR constitutive and derepressed genetic backgrounds. Two different divergent CreA sites, of which one overlaps a functional AlcR inverted repeat site, are largely responsible for alcA repression. Totally derepressed alcA expression is achieved when these two CreA sites are disrupted in addition to another single site, which overlaps the functional palindromic induction target. The fact that derepression is always associated with alcA overexpression is consistent with a competition model between AlcR and CreA for their cognate targets in the same region of the alcA promoter. Our results also indicate that the CreA repressor is necessary and sufficient for the total repression of the alcA gene.

The zinc binuclear cluster activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans.

The alcA gene which is part of the recently identified ethanol regulon, is one of the most strongly inducible genes in Aspergillus nidulans. Its transcriptional activation is mediated by the AlcR transactivator which contains a DNA-binding domain belonging to the C6 zinc binuclear cluster family. AlcR differs from the other members of this family by several features, the most striking characteristic being its binding to both symmetric and asymmetric DNA sites with the same apparent affinity. However, AlcR is also able to bind to a single site with high affinity, suggesting that unlike the other C6 proteins, AlcR binds as a monomer. In this report, we show that AlcR targets, to be functional in vivo, have to be organized as inverted or direct repeats. In addition, we show a strong synergistic activation of alcA transcription in which the number and the position of the AlcR-binding sites are crucial. The fact that the AlcR unit for in vitro binding is a single site whereas the in vivo functional unit is a repeat opens the question of the mechanism of the strong alcA transactivation. These results show that AlcR displays both in vitro and in vivo a new range of binding specificity and provides a novel example in the C6 zinc cluster protein family.

The basal level of transcription of the alc genes in the ethanol regulon in Aspergillus nidulans is controlled both by the specific transactivator AlcR and the general carbon catabolite repressor CreA.

In the A. nidulans ethanol utilization pathway, specific induction is mediated by the transactivator AlcR which is subject to strong positive autogenous regulation and activates the transcription of the two structural genes alcA and aldA. Carbon catabolite repression is mediated by CreA which represses directly the transacting gene alcR and the two structural genes. We show here that the basal expression of the alcR and alcA genes is also controlled by the two regulatory circuits, positively by the transactivator AlcR and negatively by the repressor CreA, the aldA gene being subject only to the control of the CreA repressor.

Experimental diabetic neuropathy. Effect of ganglioside treatment on axonal transport of cytoskeletal proteins.

Abnormalities in axonal transport of proteins are thought to play an important role in the pathogenesis of diabetic neuropathy. Gangliosides exert a positive action on numerous alterations in biochemistry and physiology of diabetic nerves. This study was undertaken to assess the effects of exogenous gangliosides on the axonal transport of structural proteins such as actin and tubulin in the sensory fibers of short-term (9-wk) and long-term (6-mo) diabetic rats. Adult Sprague-Dawley rats were made diabetic with a single injection of 70 mg/kg streptozocin i.p. Subgroups were injected daily with either highly purified ganglioside mixture (10 mg/kg i.p.) or saline for 1 mo, beginning either 2 or 17 wk after streptozocin injection. Age-matched rats were used as controls. Axonal transport was studied by the pulse-labeling technique. Three weeks after labeling, sciatic nerves were dissected out and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. In diabetic rats of both experimental designs, the transport rate of tubulin and actin was decreased by approximately 30% compared with control rats. Ganglioside treatment counteracted such alterations in both 9-wk and 6-mo diabetic rats. These data suggest a pharmacological effect that could be correlated with molecular interactions between integral membrane glycolipids and cytoskeletal elements.

Efficacy of ganglioside treatment in reducing functional alterations induced by vincristine in rabbit peripheral nerves.

Vincristine (VCR) administration to rabbits resulted in severe electrophysiologic alterations of peripheral nerves. Sciatic nerve conduction velocity, compound action potential (CAP) amplitude, and area under the CAP waveform were all reduced in a dose-dependent fashion. In addition, the pattern of conduction velocity of both motor and sensory fibers was altered and shifted toward slow conduction classes. Simultaneous treatment with gangliosides limited significantly the changes in electrophysiologic parameters induced by VCR. It is suggested that gangliosides be given in the clinical setting as protection for the peripheral nerves against the side effects of antiblastic therapy.

Ganglioside treatment of vincristine-induced neuropathy. An electrophysiologic study.

A 5-week treatment with vincristine (0.20 or 0.25 mg/kg, i.v., once/week) in the rabbit produced a peripheral neuropathy characterized by both morphologic and electrophysiologic alterations. In particular, electrophysiologic recordings demonstrated that the amplitude, area and conduction velocity of compound action potential (CAP) were consistently reduced in a dose-dependent manner. The simultaneous daily administration of 50 mg/kg ganglioside mixture counteracted significantly this functional impairment, area and amplitude of the monophasic CAP being maintained closer to control values. These results suggest that the loss of functional nerve fibers can be reduced, in vincristine-induced neuropathy, by ganglioside treatment.

Adaptation of sarcolemmal action potential mechanisms to chronic depolarization in denervated skeletal muscle.

Action potential properties were studied in rat extensor digitorum longus fibers, at different times after locally setting the membrane to a holding potential of -90 mV. Whereas in normal muscles holding potential duration had little effect on the action potential, the holding potential duration markedly influenced membrane excitability in the fibers previously depolarized by increasing the K+ concentration of the bathing medium. In this case, when the holding potential was prolonged from 20 to 180 s, action potential overshoot, maximum rate of rise, and maximum rate of fall increased 1.8-, 3.1-, and 1.8-fold, respectively. In the denervated muscle, overshoot and maximum rate of fall were dependent on the duration of holding potential application until denervation day 6, whereas maximum rate of rise was affected throughout the duration of this study (15 days of denervation). However, 180-s application of -90 mV holding potential elicited about a 2-fold increase of maximum rate of rise in the earlier denervation stages, and only a 1.5-fold increase at later times. These observations suggest that ultra-slow processes of Na+ conductance inactivation were less effective after 6 days of denervation. Correspondingly, extensor digitorum longus fibers acquired the ability to generate action potentials at a depolarized holding potential. The partial removal of ultra-slow Na+ inactivation after muscle denervation could substantially contribute to a general process of membrane adaptation, resulting in the capacity of voltage-dependent ion channels to operate in a condition of chronic depolarization.

Structural and biochemical alterations in the dorsal horn of the spinal cord caused by peripheral nerve lesions.

Substance P and Met-enkephalin were detected by radioimmunoassay and immunocytochemistry in the rat lumbar spinal cord. The sciatic nerve was lesioned by resecting a piece and the proximal stump was either ligated, for limiting neurite outgrowth, or intraperitoneally sutured, allowing the formation of a large neuroma. Ten days post lesioning both peptide levels dropped approximately 50% and the punctate immunoreactivity decreased in the dorsal horn. Lesioning both sciatic nerves did not accelerate nor increase the extent of peptide loss compared to monolateral lesions. Immunocytochemistry showed that after bilateral lesioning also the punctate immunoreactivity in the dorsal horn decreased less drastically. However, FRAP staining of the dorsal horn decreased according to the lesion paradigms, mono- and bilaterally with the same intensity. Therefore nerve lesions trigger the process, but the peptidergic loss seems intraspinally regulated. In addition, both kinds of abnormal neurite outgrowth similarly altered peptide levels and distribution in the spinal cord. Our data suggest that pain states related to peripheral nerve lesions may be due to opiate peptide loss rather than to neuroma.