Mitochondrial calcium flux in temperature-sensitive mutants of Drosophila

The mechanisms of mitochondrial calcium flux in normal and temperature-sensitive mutants of $\text{Drosophila}$ were investigated. Adult mitochondria from all stocks, when analysed with an oxygen electrode, gave respiration rates which exhibited normal responses to adenosine diphosphate and uncoupling agents but no stimulation by calcium. In contrast, calcium stimulates the respiration rate of normal larval mitochondria particularly those of the second instar. This is not evident in second instar mitochondria from the temperature sensitive mutants. There is a rapid accumulation of mitochondrial calcium during normal larval ontogenesis. The levels in temperature-sensitive second instar mitochondria are higher than those of any of the normal larval stages. Adult mitochondria in all cases contain very low levels of calcium. The amount of calcium taken up by mitochondria of second instar temperature-sensitive mutants is lower than that of normal. This may reflect the higher endogenous levels already present in the former. The implications of these variations in calcium metabolism for the behavioural defects of the temperature sensitive mutants is discussed.     

Genome-wide screening of yeast metal homeostasis genes involved in mitochondrial functions

Metal ions are essential for mitochondria to execute their roles. Yeast mutants that are sensitive to metals (either excess or deficiency) on non-fermentable media but not on fermentable media may carry mutations in genes that participate in metal homeostasis involving mitochondrial functions. A collection of approximately 4,800 haploid yeast deletion mutants was screened for metal ion homeostasis genes linked to mitochondrial respiration. In addition to several well-characterized metal homeostasis genes, 45 new mutants, impaired in various molecular functions, were identified on non-fermentable media that were sensitive to adscititious metals or metal deficiency. While 35 of these mutants displayed metal-sensitivity only on non-fermentable media, the remaining 10 also exhibited metal sensitivity on fermentable media, suggesting metal-sensitivity of the latter is not due to mitochondrial dysfunction. Inductively coupled plasma optical emission spectrometry (ICP-OES) was conducted for 12 mutants that were sensitive to metal excess to analyze their metal contents. Among these 12 mutants 7 were sensitive to metal excess on non-fermentable but not on fermentable media. All the seven respiration-dependent mutants displayed abnormal levels of metal ions inside mitochondria, indicative of disrupted mitochondrial metal homeostasis. This study therefore effectively identified multiple new genes involved in metal homeostasis pathways possibly pertinent to mitochondrial functions, and should be helpful for future studies to further understand their molecular roles.     

Unaltered hepatic oxidative phosphorylation and mitochondrial permeability transition in wistar rats treated with nimesulide: Relevance for nimesulide toxicity characterization

Nonsteroidal anti-inflammatory drugs have been associated with hepatotoxicity in susceptible patients. One such example is nimesulide, a preferential cyclooxygenase 2-inhibitor, widely used for the treatment of inflammation and pain. It was suggested that nimesulide could exert its hepatotoxicity by altering hepatic mitochondrial function, which was demonstrated in vitro. The objective of this study was to verify whether liver mitochondria isolated from rats treated with doses of nimesulide well above therapeutic levels possessed decreased calcium tolerance and oxidative phosphorylation, which indicates in vivo nimesulide mitochondrial toxicity. Male and female rats received nimesulide or its vehicle twice daily, for 5 days, and were killed on the seventh day for the isolation of liver mitochondria. Mitochondrial respiration, transmembrane electric potential, and calcium tolerance were characterized in all experimental groups. Nimesulide had no effect on liver mitochondrial function. Indexes of mitochondrial integrity, calcium loading capacity, and oxidative phosphorylation efficiency were unchanged between liver mitochondria from treated and control animals. In the animals tested, no evidence of degraded mitochondrial function due to nimesulide administration could be found. The results corroborate the notion that despite recognized in vitro mitochondrial toxicity, nimesulide does not cause detectable mitochondrial dysfunction in Wistar rats, even when administered in much higher concentrations than those known to have anti-inflammatory effects.     

Analysis of molting and metamorphosis in the ecdysteroid-deficient mutant L(3)3DTS of Drosophila melanogaster

The dominant temperature-sensitive mutation L(3)3^DTS (DTS-3) in Drosophila melanogaster causes lethality of heterozygotes during the third larval instar at the restrictive temperature (29°C). Temperature-shift experiments revealed two distinct temperature-sensitive periods, with lethal phases during the third larval instar (which may persist for 4 weeks) and during the late pupal stage. At 29°C mutant imaginal discs are unable to evert in situ, but did evert normally if cultured in the presence of exogenous ecdysterone or when implanted into wild-type larval hosts. The only morphologically abnormal tissue present in the lethal larvae is the ring gland, the prothoracic gland being greatly hypertrophied in third instar DTS-3 larvae. Injection of a single wild-type ring gland rescued these mutant larvae, indicating that the mutant gland is functionally, as well as morphologically, abnormal. Finally, the mutant larvae were shown to have less than 10% of the wild-type ecdysteroid levels. These results are all consistent with a proposed lesion in ecdysteroid hormone production in DTS-3 larvae. A comparison with the phenotypes of other “ecdysone-less” mutants is presented.     

Bonsaï, a ribosomal protein S15 homolog, involved in gut mitochondrial activity and systemic growth

The regulation of cellular growth is crucial in the control of cell proliferation. While most of the metabolic energy necessary to sustain growth is produced in mitochondria, the regulation of mitochondrial activity and its implications for growth have remained unexplored. Here, a gene named bonsa? is described, which is essential for normal growth in Drosophila. The Bonsa? protein bears strong homology to prokaryotic ribosomal protein S15 and localizes to mitochondria, suggesting a role in mitochondrial protein translation. Accordingly, bonsa? mutants have defective mitochondrial activity, but surprisingly, only the gut appears affected. Consistent with these observations, bonsa? is predominantly expressed in the gut. These results show that bonsa? plays a preponderant role in gut mitochondria. Although gut mitochondrial respiration is altered in bonsa? mutants, the digestive process appears normal, suggesting that a gut function other than digestion is impaired in the mutants. Cytochrome c oxidase, a respiratory chain enzyme partly encoded by the mitochondrial genome, is found to be active in bonsa? mutants. This suggests that mitochondrial translation is not abolished in the mutants. Altogether, these observations suggest that mitochondrial activity is regulated at the tissue-specific level and that this regulation has profound implications for growth and development.     

Arabidopsis thaliana organellar DNA polymerase IB mutants exhibit reduced mtDNA levels with a decrease in mitochondrial area density

Plant organelle genomes are complex and the mechanisms for their replication and maintenance remain unclear. Arabidopsis thaliana has two DNA polymerase genes, DNA polymerase IA (polIA) and polIB, that are dual targeted to mitochondria and chloroplasts and are differentially expressed in primary plant tissues. PolIB gene expression occurs at higher levels in tissues not primary for photosynthesis. Arabidopsis T‐DNA polIB mutants have a 30% reduction in relative mitochondrial DNA (mtDNA) levels, but also exhibit a 70% increase in polIA gene expression. The polIB mutant shows an increase in mitochondrial numbers but a significant decrease in mitochondrial area density within the hypocotyl epidermis, shoot apex and root tips. Chloroplast numbers are not significantly different in mesophyll protoplasts. These mutants do not have a significant difference in total dark mitorespiration levels but exhibit a difference in light respiration levels and photosynthesis capacity. Organelle‐encoded genes for components of respiration and photosynthesis are upregulated in polIB mutants. The mutants exhibited slow growth in conjunction with a decreased rate of cell expansion and other secondary phenotypic effects. Evidence suggests that early plastid development and DNA levels are directly affected by a polIB mutation but are resolved to wild‐type levels over time. However, mitochondria numbers and DNA levels never reach wild‐type levels in the polIB mutant. We propose that both polIA and polIB are required for mtDNA replication. The results suggest that polIB mutants undergo an adjustment in cell homeostasis, enabling them to maintain functional mitochondria at the cost of normal cell expansion and plant growth.     

Extrachromosomal oligomycin-resistant mutants of the petite-negative yeast Kluyveromyces lactis. Properties of mitochondrial ATPase and cross-resistance to inhibitors of phosphoryl transfer reactions

Summary The mitochondrial ATPase from oligomycin-resistant mutants which map on different regions of an extrachromosomal DNA (01 and 011 class mutants) showed an increased resistance to oligomycin and venturicidin when assayed in vitro as compared to the sensitive strains.The resistance to oligomycin of the isolated mitochondrial ATPase from 01 class mutants was higher than that of the 011 class mutants.Cross resistance of the oligomycin-resistant mutants to the antibiotics peliomycin and ossamycin, which also inhibit phosphoryl transfer reactions in mitochondria (Walter et al., 1967), was observed, 01 mutants being more resistant to ossamycin than 011 class mutants. At the concentrations of peliomycin studied, no difference in sensitivity among both groups of oligomycin-resistant mutants could be detected.Mitochondrial respiration and isolated mitochondrial ATPase activity are sensitive to venturicidin, suggesting that the previously observed (Brunner et al., 1977) in vivo venturicidin resistance of K. lactis is probably due to an impairment of the influx of the drug at the level of the plasma membrane.     

2,3-butanedione monoxime unmasks Ca(2+)-induced NADH formation and inhibits electron transport in rat hearts

We used 2,3-butanedione monoxime (BDM) to suppress work by the perfused rat heart and to investigate the effects of calcium on NADH production and tissue energetics. Hearts were perfused with buffer containing BDM and elevated perfusate calcium to maintain the rates of cardiac work and oxygen consumption at levels similar to those of control perfused hearts. BDM plus calcium hearts displayed higher levels of NADH surface fluorescence, indicating calcium activation of mitochondrial dehydrogenases. These hearts, however, displayed 20% lower phosphocreatine levels. BDM suppressed the rates of state 3 respiration of isolated mitochondria. Uncoupled respiration was suppressed to a lesser degree, and the state 4 respiration rates were not affected. Double-inhibitor experiments with liver mitochondria using BDM and carboxyatractyloside (CAT) were used to identify the site of inhibition. BDM at low levels (0-5 mM) suppressed respiration. In the presence of CAT at levels that inhibit respiration by 60%, low levels of BDM were without effect. Because these effects were not additive, BDM does not inhibit adenine nucleotide transport. This was supported by an assay of adenine nucleotide transport in liver mitochondria. BDM did not inhibit ATP hydrolysis by submitochondrial particles but strongly suppressed reversed electron transport from succinate to NAD(+). Oxidation of NADH by submitochondrial particles was inhibited by BDM but oxidation of succinate was not. We conclude that BDM inhibits electron transport at site 1.     

MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria

In the yeast Saccharomyces cerevisiae, mitochondria are elongated organelles which form a reticulum around the cell periphery. To determine the mechanism by which mitochondrial shape is established and maintained, we screened yeast mutants for those defective in mitochondrial morphology. One of these mutants, mmm1, is temperature- sensitive for the external shape of its mitochondria. At the restrictive temperature, elongated mitochondria appear to quickly collapse into large, spherical organelles. Upon return to the permissive temperature, wild-type mitochondrial structure is restored. The morphology of other cellular organelles is not affected in mmm1 mutants, and mmm1 does not disrupt normal actin or tubulin organization. Cells disrupted in the MMM1 gene are inviable when grown on nonfermentable carbon sources and show abnormal mitochondrial morphology at all temperatures. The lethality of mmm1 mutants appears to result from the inability to segregate the aberrant-shaped mitochondria into daughter cells. Mitochondrial structure is therefore important for normal cell function. Mmm1p is located in the mitochondrial outer membrane, with a large carboxyl-terminal domain facing the cytosol. We propose that Mmm1p maintains mitochondria in an elongated shape by attaching the mitochondrion to an external framework, such as the cytoskeleton.     

Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms

We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.     

Isolation and biochemical analysis of a temperature-sensitive scarlet eye color mutant of Drosophila melanogaster

Six new EMS-induced scarlet mutants were selected. Four of these were partially pigmented, with xanthommatin levels ranging from 12% to 45% of normal. In one (st ^754ts), pigment production was temperature sensitive; the level of xanthommatin changed from less than 10% of normal at 29 C to more than 70% at 18 C. In all of the new mutants tested, the level of early pupal 3-hydroxykynurenine was as low as low as that in st ¹. Thus reduced larval accumulation of this metabolite also appears to be a characteristic feature of scarlet mutants. Temperature-pulse and temperature-shift experiments were carried out with st ^754ts to determine the temperature-sensitive period for the scarlet gene during development. The major sensitive period commenced prior to the onset of pigmentation and was over before adult emergence. Thus the initiation of xanthommatin synthesis is not brought about by the activation of the scarlet gene. In similar experiments carried out with a temperature-sensitive white mutant (w ^bl), a similar temperature-sensitive period was obtained.This work was supported by Grant D2 75/15248 from the Australian Research Grants Committee and also by Grant GB 27599 from The National Science Foundation to Professor M. M. Green.     

Mitochondrial single-stranded DNA-binding protein is required for mitochondrial DNA replication and development in Drosophila melanogaster

The discovery that several inherited human diseases are caused by mtDNA depletion has led to an increased interest in the replication and maintenance of mtDNA. We have isolated a new mutant in the lopo (low power) gene from Drosophila melanogaster affecting the mitochondrial single-stranded DNA-binding protein (mtSSB), which is one of the key components in mtDNA replication and maintenance. lopo(1) mutants die late in the third instar before completion of metamorphosis because of a failure in cell proliferation. Molecular, histochemical, and physiological experiments show a drastic decrease in mtDNA content that is coupled with the loss of respiration in these mutants. However, the number and morphology of mitochondria are not greatly affected. Immunocytochemical analysis shows that mtSSB is expressed in all tissues but is highly enriched in proliferating tissues and in the developing oocyte. lopo(1) is the first mtSSB mutant in higher eukaryotes, and its analysis demonstrates the essential function of this gene in development, providing an excellent model to study mitochondrial biogenesis in animals.     

Decreased JH biosynthesis is related to precocious metamorphosis in recessive trimolter (rt) mutants of the silkworm, Bombyx mori

In recessive trimolter (rt) mutants of the silkworm, Bombyx mori, that have four larval instars rather than five larval instars of normal B. mori, a decrease after a small increase in the hemolymph ecdysteroid titer during the early stages of the last (fourth) larval instar appeared to be a prerequisite for larvae to undergo precocious metamorphosis. The present study was carried out to investigate the possible mechanism underlying this decrease in the ecdysteroid titer. It was found that juvenile hormone (JH) biosynthetic activity of the corpora allata (CA) increased during the first day of the last larval instar, but its absolute JH biosynthesis activity was relatively lower compared to that of normal fourth-instar larvae in tetramolters. This lowered JH biosynthetic activity appeared to be related to a decrease in prothoracic gland ecdysteroidogenesis during the second day of the last instar, because hydroprene application prevented this decrease in prothoracic gland ecdysteroidogenesis, leading to the induction of a supernumerary larval molt. The in vitro incubation of prothoracic glands with hydroprene showed that hydroprene did not directly exert its action on prothoracicotropic hormone (PTTH) release. Further study showed that the application of hydroprene enhanced the competency of the glands to respond to PTTH. From these results, it was supposed that the lowered JH biosynthesis of the CA during the first day of last instar in rt mutants was related to decreased ecdysteroidogenesis in the prothoracic glands during the second day, thus playing a role in leading to precocious metamorphosis.     

Calcium-induced contraction of sarcomeres changes the regulation of mitochondrial respiration in permeabilized cardiac cells

The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyocytes and myocardial fibers, and in their 'ghost' preparations (after selective myosin extraction). Up to 3 microm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and a significant decrease in the apparent K(m) for exogenous ADP and ATP in the kinetics of mitochondrial respiration. The V(max) of respiration showed a moderate (50%) increase, with an optimum at 0.4 microm free calcium and a decrease at higher calcium concentrations. At high free-calcium concentrations, the direct flux of ADP from ATPases to mitochondria was diminished compared to that at low calcium levels. All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in 'ghost' preparations after the selective extraction of myosin. Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition to the metabolic signalling via the creatine kinase system.     

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.     

Partial protection against erucoyl-carnitine inhibition in hamster brown-adipose-tissue mitochondria is due to high CoA levels: a comparison with rat brown-adipose-tissue mitochondria

Brown-adipose-tissue mitochondria isolated from golden hamsters were found to contain more CoA per mg protein than rat brown-fat mitochondria, and after incubation with erucoyl-carnitine, a higher free CoA level remained, than in rat mitochondria. In accordance with the suggestion (Alexson et al. (1985) Biochim. biophys. Acta 834, 149-158) that the inhibitory effect of erucoyl-carnitine on brown-fat mitochondrial respiration is entirely due to CoA sequestration, hamster mitochondria (with more CoA) were less sensitive to erucoyl inhibition than were rat mitochondria. Thus, increased mitochondrial CoA levels may augment the ability of animals to withstand the detrimental effects of a high erucoyl ester content of the diet.     

Study of temperature-sensitive mutations in the virilis group of Drosophila. 4. Phenogenetics of a temperature-sensitive mutation affecting the development of imaginal discs in Drosophila virilis Sturt]

The phenogenetics of a new temperature-sensitive mutation diskless-ts (dl-ts, 2nd chromosome) has been studied in D. virilis. The rearing of larvae from the 1st instar at the temperature 31 degrees resulted in the complete or partial arrest of the development of imaginal discs and, consequently, the death of larvae prior to the pupation, at the prepupa stage. The change of temperature from 25 to 31 degrees during the second half of development in the 3rd instar affects the differentiation of imaginal discs. Some organs differentiate completely or partially (eyes, wings, legs) whereas the rest do not develop at all. The sensitive period embraces the whole larval development till the beginning of pupation.     

线粒体ATP敏感钾通道与钙激活钾通道激活对正常与缺血脑线粒体通透性转变的影响

目的：明确线粒体ATP敏感钾通道与钙激活钾通道对正常和缺血脑线粒体渗透性转变的作用。方法：实验采用分光光度法,在分离的线粒体上分别观察两种线粒体钾通道激动剂对正常与缺血脑线粒体肿胀的影响。结果：在正常脑线粒体,diazoxide与NSl619能有效抑制由钙诱导的线粒体氏20下降,但其效应可被atractyloside所阻断。与正常相比,缺血损伤后的脑线粒体在钙离子诱导下线粒体A520下降较快,diazoxide与NS1619仍可抑制由钙诱导的线粒体A520下降,其作用同样为atractykxside所阻断。结论：线粒体ATP敏感钾通道与钙激活钾通道激活在离体条件均具有保护脑线粒体的作用,其作用可能是通过影响线粒体通透性转变而实现。     

An efficient genetic screen in Drosophila to identify nuclear-encoded genes with mitochondrial function

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.     

Illumination with blue light reactivates respiratory activity of mitochondria inhibited by nitric oxide, but not by glycerol trinitrate

Nitric oxide (NO) is known to inhibit mitochondrial respiration reversibly. This study aimed at clarifying whether low level illumination at specific wavelengths recovers mitochondrial respiration inhibited by NO and glycerol-trinitrate (GTN), a clinically used NO mimetic. NO fully inhibited respiration of liver mitochondria at concentrations occurring under septic shock. The respiration was completely restored by illumination at the wavelength of 430 nm while longer wavelengths were less effective. GTN inhibited mitochondrial respiration though the efficiency of GTN was lower compared to NO concentrations observed in sepsis models. However, GTN inhibition was absolutely insensitive to illumination regardless of wavelength used. Our data show that visible light of short wavelengths efficiently facilitates the recovery of mitochondria inhibited by NO-gas at the levels generated under septic conditions. The inhibition of mitochondrial respiration by GTN is not sensitive to visible light, suggesting an inhibition mechanism other that NO mediation.