Growth and patterning are evolutionarily dissociated in the vestigial wing discs of workers of the red imported fire ant, Solenopsis invicta

Over the last decade, it has become clear that organismal form is largely determined by developmental and evolutionary changes in the growth and pattern formation of tissues. Yet, there is little known about how these two integrated processes respond to environmental cues or how they evolve relative to one another. Here, we present the discovery of vestigial wing imaginal discs in worker larvae of the red imported fire ant, Solenopsis invicta. These vestigial wing discs are present in all worker larvae, which is uncommon for a species with a large worker size distribution. Furthermore, the growth trajectory of these vestigial discs is distinct from all of the ant species examined to date because they grow at a rate slower than the leg discs. We predicted that the growth trajectory of the vestigial wing discs would be mirrored by evolutionary changes in their patterning. We tested this prediction by examining the expression of three patterning genes, extradenticle, ultrabithorax, and engrailed, known to underlie the wing polyphenism in ants. Surprisingly, the expression patterns of these three genes in the vestigial wing discs was the same as those found in ant species with different worker size distributions and wing disc growth than fire ants. We conclude that growth and patterning are evolutionarily dissociated in the vestigial wing discs of S. invicta because patterning in these discs is conserved, whereas their growth trajectories are not. The evolutionary dissociation of growth and patterning may be an important feature of gene networks that underlie polyphenic traits.     

Isolation and characterization of mutants defective in the cyanide-insensitive respiratory pathway of Pseudomonas aeruginosa.

The branched respiratory chain of Pseudomonas aeruginosa contains at least two terminal oxidases which are active under normal physiological conditions. One of these, cytochrome co, is a cytochrome c oxidase which is completely inhibited by concentrations of the respiratory inhibitor potassium cyanide as low as 100 microM. The second oxidase, the cyanide-insensitive oxidase, is resistant to cyanide concentrations in excess of 1 mM as well as to sodium azide. In this work, we describe the isolation and characterization of a mutant of P. aeruginosa defective in cyanide-insensitive respiration. This insertion mutant was isolated with mini-D171 (a replication-defective derivative of the P. aeruginosa phage D3112) as a mutagen and by screening the resulting tetracycline-resistant transductants for the loss of ability to grow in the presence of 1 mM sodium azide. Polarographic studies on the NADH-mediated respiration rate of the mutant indicated an approximate 50% loss of activity, and titration of this activity against increasing cyanide concentrations gave a monophasic curve clearly showing the complete loss of cyanide-insensitive respiration. The mutated gene for a mutant affected in the cyanide-insensitive, oxidase-terminated respiratory pathway has been designated cio. We have complemented the azide-sensitive phenotype of this mutant with a wild-type copy of the gene by in vivo cloning with another mini-D element, mini-D386, carried on plasmid pADD386. The complemented cio mutant regained the ability to grow on medium containing 1 mM azide, titration of its NADH oxidase activity with cyanide gave a biphasic curve similar to that of the wild-type organism, and the respiration rate returned to normal levels. Spectral analysis of the cytochrome contents of the membranes of the wild type, the cio mutant, and the complemented mutant suggests that the cio mutant is not defective in any membrane-bound cytochromes and that the complementing gene does not encode a heme protein.     

Aldehyde oxidase distribution in the imaginal discs of some diptera

Summary In the imaginal discs ofMusca domestica, Drosophila melanogaster, D. simulans, D. hydei, andZaprionus spec. the enzyme aldehyde oxidase (AO) appeared in a clear-cut pattern. In the leg and eye-antennal discs of these species this pattern shows a high degree of conformity, while that of the wing and haltere discs is species-specific.No aldehyde oxidase activity was detected in the imaginal discs ofCalliphora erythrocephala, Phormia regina orLucilia cuprina, but the discs of these species are characterized by grossly similar patterns of 5-nucleotidase. Since the other species studied lack this enzyme, the two enzymes may perform similar functions in the morphogenesis of the discs.The coincidence of the sharp boundary of the AO pattern in the leg and wing discs ofD. melanogaster with the boundary between the anterior and posterior disc compartments gives a strong indication for the existence of analogous compartments in other discs showing a similar sharply bounded AO pattern. Compartmentalization may be considered a general phenomenon which occurs in discs of all segments and is not restricted toD. melanogaster. From the changes in the AO pattern during disc development it can be deduced that the localisation of this enzyme is regulated by supracellular determination involving positional information.     

Regulation of mitochondrial membrane assembly in Neurospora crassa. Transient expression of a respiratory mutant phenotype.

Cultures of mutant cni-1, a chromosomal mutant of Neurospora crassa, undergo a marked change in respiratory properties as the age of the culture increases. Early log phase cultures have a high level of respiration that is insensitive to inhibition by cyanide or antimycin A. Late log and stationary phase cultures have reduced rates of respiration. A high percentage of this respiration is inhibited by cyanide. Mitochondria from early log phase cni-1 have an excess of cytochrome c and little or no detectable cytochrome aa3. Mitochondria from late log and stationary phase cultures have levels of c-, b-, and a-type cytochromes that are not significantly different in concentration from those found in wild type cells. The cytochrome aa3 content and the cytochrome oxidase activity of cni-1 mitochondria increase 5- to 10-fold as the age of the culture increases. Mitochondria from early log phase cells of cni-1 synthesize only polypeptides of apparent molecular weights 7,000 to 10,000 and donot synthesize any of the mitochondrial components of cytochrome oxidase. Mitochondria from late log and stationary phase cells synthesize the normal complement of mitochondrial translation products including the mitochondrial components of cytochrome oxidase. The assembly of cytochrome oxidase is likely due to the availability of the mitochondrially synthesized components of the enzyme. The regulation of mitochondrial translation in the cni-1 mutant is independent of the nutrient content of the growth medium and is due to the accumulation or depletion of some component within the cell.     

Allelic interactions at the engrailed locus of Drosophila: engrailed protein expression in imaginal discs

Many embryonic lethal engrailed (enlethal) mutations are known to partially complement the cuticular defects of the original engrailed mutation, en1. To explore the nature of this complementation, the adult phenotypes of several different en1/enlethal transheterozygotes were compared with the corresponding patterns of engrailed protein expression in third larval instar imaginal discs (determined by immunofluorescence). Transheterozygotes of en1 and deletions of the locus (enDf) typically show slight complementation in the adult cuticle. The pattern of engrailed protein expression in some en1/enDf wing discs is indistinguishable from en1 homozygotes, but in others the pattern is nearly normal. en1/enDf leg discs appear to express engrailed protein normally. Transheterozygotes of en1 and EMS-induced, cytologically normal enlethal alleles have almost normal adult cuticle phenotypes and also exhibit normal patterns of engrailed protein expression in all of the thoracic imaginal discs. Surprisingly, the intensity of anti-engrailed staining in these discs is elevated relative to that in wild type. en2 is an unusual lethal allele in that it does not complement either the en1 adult cuticle phenotype or the protein expression pattern in imaginal discs. Moreover, the cytologically normal enlethal alleles also complement en2, at least partially. Both wing and leg imaginal discs from en2/enlethal transheterozygotes show abnormal patterns of engrailed protein expression. These results are discussed in the context of an autoregulatory model for engrailed regulation.     

Benzoylphenyl ureas inhibit chitin synthesis without interfering with amino sugar uptake in imaginal wing discs of Plodia interpunctella (Hübner)

We tested the hypothesis that the inhibition of chitin synthesis by benzoylphenyl ureas could be explained by their effect on the uptake of GlcNAc into chitin. Our test system consisted of organ cultures of wing imaginal discs from Plodia interpunctella. These wing discs synthesize chitin in response to 20-hydroxyecdysone or RH 5849, a non-steroidal ecdysteroid mimic. Two benzoylphenyl ureas, diflubenzuron and teflubenzuron, inhibited ecdysteroid-dependent chitin synthesis in the wing discs. However, although chitin synthesis was inhibited, there was no corresponding diminution of amino sugar uptake by the imaginal discs. In another experiment 20-hydroxyecdysone stimulated uptake of two sugars, 2-deoxy-D-glucose and 3-O-methyl-D-glucose, which are not synthesized into chitin. Transport of these non-metabolized sugars was unaffected by the inhibitors. In an additional test of the effects on precursor transport, the action of ecdysone (alpha-ecdysone) was examined. Ecdysone stimulated amino sugar uptake, but not chitin synthesis. Neither diflubenzuron nor teflubenzuron inhibited ecdysone-dependent precursor transport. Finally, we examined ecdysteroid-induced uptake of amino sugars by an imaginal disc derived cell line IAL-PID2. In this case, also, GlcNAc transport was not inhibited significantly by either diflubenzuron or teflubenzuron. From these observations we conclude that inhibition of uptake of amino sugars does not account for the ability of teflubenzuron and diflubenzuron to inhibit chitin synthesis in P. interpunctella wing discs.     

Respiratory terminal oxidases in the facultative chemoheterotrophic and dinitrogen fixing cyanobacterium Anabaena variabilis strain ATCC 29413: characterization of the cox2 locus

Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process. Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains. We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation. Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases. One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized. Up-regulation of Cox2 was corroborated by Northern and primer extension analyses. Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically. In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions. Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.     

Methotrexate: studies on the cellular metabolism. I. Effect on mitochondrial oxygen uptake and oxidative phosphorylation

Effect of methotrexate (MTX) on mitochondrial oxygen uptake, oxidative phosphorylation and on the activity of several enzymes linked to respiratory chain was studied. MTX was able to inhibit state III respiration activated by ADP and to decrease the respiratory coefficient with the substrates alpha-ketoglutarate and glutamate; these effects became pronounced when mitochondria were pre-incubated with MTX for 10 min. No effect was observed on ATPase activity of undamaged or broken mitochondria; the same was true for NADH-oxidase, NADH-dehydrogenase, NADH-cytochrome c reductase, succinate oxidase, and cytochrome c oxidase activity. The effect on the steady-state of cytochrome b, as well as, the inhibitory effect on state III of respiration with NAD+-linked substrates, offers a reasonable possibility to suggesting that the inhibition site of MTX could be in a place anterior to cytochrome b region, and not linked to respiratory chain.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.     

Energy transduction in photosynthetic bacteria. X. Composition and function of the branched oxidase system in wild type and respiration deficient mutants of Rhodopseudomonas capsulata.

The respiratory chain of Rhodopseudomonas capsulata, strain St. Louis and of two respiration deficient mutants (M6 and M7) has been investigated by examining the redox and spectral characteristics of the cytochromes and their response to substrates and to specific respiratory inhibitors. Since the specific lesions of M6 and M7 have been localized on two different branches of the multiple oxidase system of the wild type strain, the capability for aerobic growth of these mutants can be considered as a proof of the physiological significance of both branched systems "in vivo". Using M6 and M7 mutants the response of the branched chain to respiratory inhibitors could be established. Cytochrome oxidase activity, a specific function of an high potential cytochrome b (E'0 = +413 mV) is sensitive to low concentrations of KCN (5-10(-5) M); CO is a specific inhibitor of an alternative oxidase, which is also inhibited by high concentrations of KCN (10(-3) M). Antimycin A inhibits preferentially the branch of the chain affected by low concentrations of cyanide. Redox titrations and spectral data indicate the presence in the membrane of three cytochromes of b type (E'0 = +413, +260, +47 vM) and two cytochromes of c type (E'0 = +342, +94 mV). A clear indication of the involvement in respiration of cytochrome b413, cytochrome c342 and cytochrome b47 has been obtained. Only 50% of the dithionite reducible cytochrome b can be reduced by respiratory substrates also in the presence of high concentrations of KCN or in anaerobiosis. The presence and function of quinones in the respiratory electron transport system has been clearly demonstrated. Quinones, which are reducible by NADH and succinate to about the same extent can be reoxidized through both branches of the respiratory chain, as shown by the response of their redox state to KCN. The possible site of the branching of the electron transport chain has been investigated comparing the per cent level of reduction of quinones and of cytochromes b and c as a function of KCN concentrations in membranes from wild type and M6 mutants cells. The site of the branching has been localized at the level of quinones-cytochrome b47. A tentative scheme of the respiratory chains operating in Rhodopseudomonas capsulata, St. Louis and in the two respiration deficient mutants, M6 and M7 is presented.     

Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6-100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco2, dco3, and dco18) in heteroallelic combinations and one allele (dco3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dcole88), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a "discless" phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7-8 and 12-14 days AEL. In c43hs1, another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3/dco18 and c43hs1 wing discs, although not in dco2/dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.     

Ecdysteroid concentration inducing cell proliferation brings about the imaginal differentiation in the wing disc of Bombyx mori in vitro

This study was undertaken to evaluate the range of 20-hydroxyecdysone (20HE) concentrations which induce cell proliferation and imaginal differentiation in lepidopteran wing discs in vitro . Wing discs were cultured in medium containing various doses of 20HE. During imaginal differentiation in vitro , wing discs were observed histologically and the number of mitosis was counted every day. Wing discs differentiated adult features in medium containing 0.02–0.2 μg/mL 20HE, and these doses also increased the number of mitosis in disc cells. Wing discs developed the same in vitro as they do in vivo . The concentration of 20HE over 0.2 μg/mL inhibited both mitosis and imaginal differentiation. Cell proliferation, cuticle deposition and tissue elongation were successively observed in vitro the same as observed in vivo . These results suggest that a moderate concentration of ecdysteroid can induce cell proliferation followed by imaginal differentiation.     

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : V. CYTOCHROME OXIDASE ACTIVITY IN THE EGGS OF ARBACIA PUNCTULATA

1. An enzyme capable of oxidizing reduced cytochrome c (i.e. a cytochrome oxidase) has been obtained from Arbacia eggs. In 0.02 M hydroquinone, the cytochrome oxidase was half activated at a cytochrome c concentration of approximately 4 x 10^–6 M. The concentration of the cytochrome oxidase was found to be nearly the same in unfertilized and fertilized eggs, the amount of the enzyme—as measured by means of its activity toward cytochrome c as a representative substrate—being more than sufficient to account for the highest rate of oxygen utilization yet observed in the intact, living, fertilized eggs, and of the same order as that in certain rat tissues. 2. The Arbacia cytochrome oxidase was strongly inhibited by carbon monoxide in the dark, the inhibition being almost completely reversed by light. The inhibition constant was not greatly altered by variation in the concentration of cytochrome c or the concentration of hydroquinone used as reductant for the cytochrome c, having a value of 3 to 5 under the conditions used. The inhibition constant was about 2 with p-phenylenediamine as reductant for the cytochrome c, but apparently had the surprisingly low value of about 0.5 with 0.02 M cysteine as reductant. 3. The cytochrome oxidase was completely inhibited by sufficiently high concentrations of sodium cyanide, sodium azide, and sodium sulfide. It was also completely inhibited in 0.6 M sodium chloride. It was not inhibited by two inhibitors of copper containing enzymes, 8-hydroxyquinoline and sodium diethyldithiocarbamate. It was also not significantly inhibited by 2,4-dinitrothymol, 2,4-dinitro-o-cyclohexylphenol, phenylurethane, 5-isoamyl-5-ethylbarbituric acid, or iodoacetic acid. 4. Quantitative examination of the fertilized eggs showed that cytochrome c, if present at all, occurred in a concentration of less than 2 micrograms per gram of wet fertilized Arbacia eggs. On the basis of these data and those of Fig. 2, above, it seems safe to conclude that cytochrome c cannot carry a significant fraction of the oxygen consumption of fertilized Arbacia eggs. It was also found that, in contrast to similar preparations from certain other animal tissues, the Arbacia cytochrome oxidase preparation displayed no succinic dehydrogenase activity when tested manometrically in the presence of excess cytochrome c. 5. Extending previously reported (3) experiments with other inhibitors, the effects of sodium azide and sodium sulfide on the respiration and cell division of fertilized Arbacia eggs were determined, the eggs being initially exposed to the reagents 30 minutes after fertilization at 20°C. With either reagent cleavage was completely blocked by a concentration of reagent which reduced the respiration to approximately 50 per cent of the normal level. 6. On the basis of certain theoretical considerations regarding the possible mechanism of action of cyanide and other respiratory inhibitors it is suggested that a fraction of the respiration apparently concerned with supplying energy for division processes in the fertilized Arbacia egg may be keyed into the respiratory cycle through a carrier having a somewhat higher potential than those which carry the larger portion of the egg respiration. The theory is also employed in an effort to resolve a number of hitherto apparently paradoxical observations regarding the effects of cyanide, azide, and carbon monoxide on cell respiration.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. III: Role of the peripodial membrane

We determined the contribution of the peripodial membrane to chitin synthesis in cultured wing imaginal discs of Spodoptera frugiperda. This was accomplished by examining chitin synthesis in vitro in intact imaginal discs, in the peripodial membrane, and in imaginal discs in which the peripodial membrane had been injured. Chitin synthesis in peripodial membrane-deprived imaginal discs, peripodial membrane injured imaginal discs, and peripodial membrane fragments was assessed by measuring incorporation of [¹⁴C]GlcNAc after treatment with 20-hydroxyecdysone in tissue culture. Removing or injuring the peripodial membrane resulted in a marked decrease in ecdysteroid-dependent chitin synthesis in these wing discs compared with intact wing discs. In addition, a break in the ecdysteroid treatment of 4 h reduced chitin synthesis in the wing discs substantially. These biochemical experiments were supplemented with ultrastructural and immunocytochemical approaches. A wheat germ agglutinin colloidal gold complex was used to visualize the presence of chitin synthesized by wing discs including the peripodial membrane. These experiments confirmed the importance of an intact peripodial membrane for optimal production of cuticle by the wing pouch. Our results demonstrate that for opti-ma1 production of chitin in tissue culture, wing discs must be treated with 20-hydroxyecdysone for an uninterrupted period of 48 h, and the peripodial membrane of these imaginal discs must be present and uninjured. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Studies on the effects of copper deficiency on rat liver mitochondria. II. Effects on oxidative phosphorylation

Effects of dietary copper deficiency in rats on respiratory enzymes of isolated rat liver mitochondria have been studied. After 2 weeks of Cu-depletion, cytochrome c oxidase (EC 1.9.3.1) activity had declined by 42% and between 4 and 8 weeks exhibited between 20 and 25% of the activity of control mitochondria. Activities of NADH cytochrome c reductase (EC 1.6.99.3) and succinate cytochrome c reductase (EC 1.3.99.1), were unaffected initially but declined by 32 and 46%, respectively, after 8 weeks of Cu-depletion. After 4 weeks there was a significant (34%) decline in succinate supported state 3 respiration with only a modest (18%) decline in state 4 respiration. The ADP:O ratio was unaffected by Cu-depletion after 6 and 8 weeks of dietary Cu-restriction. State 3 respiration was significantly reduced after 6 weeks when glutamate/malate or beta-hydroxybutyrate were used as substrates, whereas state 4 respiration and ADP:O ratios were unaffected. The fall in state 3 respiration was of sufficient magnitude at 8 weeks to cause a significant decline in the respiratory control ratio with all substrates. Comparisons between the relative activities of cytochrome c oxidase and reductase activities in Cu-deficient preparations, the relatively specific effect of the deficiency on state 3 respiration with all substrates tested and the ability to increase significantly oxygen consumption in excess of maximal state 3 respiration by the uncoupler 2,4-dinitrophenol suggest that the defect in Cu-deficient mitochondria cannot be attributed solely to the decreased activity of cytochrome c oxidase.     

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.     

Suppressors of the vestigial mutant phenotype increase the level of expression of the gene

Suppressor genes of the vestigial phenotype have been isolated in a wild-type population. These suppressors have an effect on different wing mutants and are allele-specific in the case of vestigial . In a vg^BG background they produced overgrowth of the imaginal wing disc. They also induce cell death in the wild-type strain and alter the distribution of cell death in the mutant strain. Expression of vestigial is increased in the wing disc only. Hypotheses formed to determine the nature of these suppressors are in favor of a direct interaction between these genes and vestigial .     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

Cyanide- and hydroxamate-resistant respiration in Neurospora crassa.

Strain inl-89601 of Neurospora crassa respires exclusively by means of the mitochondrial cytochrome chain. The respiration of this strain is entirely inhibited by cyanide or antimycin A, the classical inhibitors of cytochrome chain respiration. When this strain was grown in the presence of chloramphenicol, however, two additional terminal oxidases were detected. One of these oxidases is inhibited by substituted hydroxamic acids and has been described previously. The second oxidase was not inhibited by cyanide or hydroxamic acid but was inhibited by azide in the presence of both cyanide and hydroxamic acid. This azide-sensitive respiration was due to a single respiratory pathway with a Ki for azide of 200 micrometer. A small amount of azide-sensitive respiration was detected in mitochondrial fractions obtained from chloramphenicol-treated cells, and it is likely that the azide-sensitive oxidase is localized in the mitochondrion. The determinants for the azide-sensitive and hydroxamate-sensitive oxidases segregate in a Mendelian manner in crosses and are either unlinked or not closely linked to each other.