Structural organization of the human short-chain acyl-CoA dehydrogenase gene

Short-chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme that catalyzes the initial reaction in short-chain fatty acid β-oxidation. Defects in the SCAD enzyme are associated with failure to thrive, often with neuromuscular dysfunction and elevated urinary excretion of ethylmalonic acid (EMA). To define the genetic basis of SCAD deficiency and ethylmalonic aciduria in patients, we have determined the sequence of the complete coding portion of the human SCAD gene (ACADS) and all of the intron-exon boundaries. The SCAD gene is approximately 13 kb in length and consists of 10 exons. Four polymorphic sites have previously been detected by sequencing of cDNA from fibroblasts of patients excreting elevated amounts of EMA. Three of these polymorphisms (321T/C, 990C/T, 1260G/C) are silent variants, while a 625G/A polymorphism results in an amino acid replacement and has been shown to be associated with ethylmalonic aciduria. From analysis of 18 unrelated Danish families, we show that the four SCAD gene polymorphisms constitute five allelic variants of the SCAD gene, and that the 625A variant together with the less frequent variant form of the three other polymorphisms (321C, 990T, 1260C) constitutes an allelic variant with a frequency of 22% in the general Danish population. Using fluorescence in-situ hybridization, we confirm the localization of the human SCAD gene to the distal part of Chromosome (Chr) 12 and suggest that the SCAD gene is a single-copy gene. The evolutionary relationship between SCAD and five other members of the acyl-CoA dehydrogenase family was investigated by two independent approaches that gave similar phylogenetic trees. Received: 10 April 1997 / Accepted: 8 August 1997     

The catalytic mechanism of Pseudomonas cytochrome c peroxidase

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with H₂O₂ was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.     

Effect of (a)synchronous light fluctuation on diversity,functional and structural stability of a marine phytoplankton metacommunity

Disentangling the mechanisms that maintain the stability of communities and ecosystem properties has become a major research focus in ecology in the face of anthropogenic environmental change. Dispersal plays a pivotal role in maintaining diversity in spatially subdivided communities, but only a few experiments have simultaneously investigated how dispersal and environmental fluctuation affect community dynamics and ecosystem stability. We performed an experimental study using marine phytoplankton species as model organisms to test these mechanisms in a metacommunity context. We established three levels of dispersal and exposed the phytoplankton to fluctuating light levels, where fluctuations were either spatially asynchronous or synchronous across patches of the metacommunity. Dispersal had no effect on diversity and ecosystem function (biomass), while light fluctuations affected both evenness and community biomass. The temporal variability of community biomass was reduced by fluctuating light and temporal beta diversity was influenced interactively by dispersal and fluctuation, whereas spatial variability in community biomass and beta diversity were barely affected by treatments. Along the establishing gradient of species richness and dominance, community biomass increased but temporal variability of biomass decreased, thus highest stability was associated with species-rich but highly uneven communities and less influenced by compensatory dynamics. In conclusion, both specific traits (dominance) and diversity (richness) affected the stability of metacommunities under fluctuating conditions.     

A Histidine-rich Linker Region in Peptidylglycine α-Amidating Monooxygenase Has the Properties of a pH Sensor

Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.     

Programmed cell death of retinal cone bipolar cells is independent of afferent or target control

Programmed cell death contributes to the histogenesis of the nervous system, and is believed to be modulated through the sustaining effects of afferents and targets during the period of synaptogenesis. Cone bipolar cells undergo programmed cell death during development, and we confirm that the numbers of three different types are increased when the pro-apoptotic Bax gene is knocked out. When their cone afferents are selectively eliminated, or when the population of retinal ganglion cells is increased, however, cone bipolar cell number remains unchanged. Programmed cell death of the cone bipolar cell populations, therefore, may be modulated cell-intrinsically rather than via interactions with these synaptic partners.     

Female Remating Behavior in a Lekking Moth

In polyandrous species, male reproductive success will at least partly be determined by males' success in sperm competition. To understand the potential for post‐mating sexual selection, it is therefore important to assess the extent of female remating. In the lekking moth Achroia grisella, male mating success is strongly determined by female choice based on the attractiveness of male ultrasonic songs. Although observations have indicated that some females will remate, only little is known about the level of sperm competition. In many species, females are more likely to remate if their first mating involved an already mated male than if the first male was virgin. Potentially, this is because mated males are less well able to provide an adequate sperm supply, nutrients, or substances inhibiting female remating. This phenomenon will effectively reduce the strength of pre‐copulatory sexual selection because attractive males with high mating success will be more susceptible to sperm competition. We therefore performed an experiment designed both to provide a more precise estimate of female remating probability and simultaneously to test the hypothesis that female remating is influenced by male mating history. Overall, approximately one of five females remated with a second male. Yet, although females mated to non‐virgin males were somewhat more prone to remate, the effect of male mating history was not significant. The results revealed, however, that heavier females were more likely to remate. Furthermore, we found that females' second copulations were longer, suggesting that, in accordance with theory, males may invest more sperm in situations with an elevated risk of sperm competition.     

Sexual reproduction in the phyllodioicous bryophyte Homalothecium lutescens (Hedw.) H.Rob. in relation to habitat age,growth conditions and genetic variation

Sporophyte production and female fertility were investigated in seventeen calcareous grassland demes of the moss Homalothecium lutescens (Hedw.) H.Rob. on the Baltic island of Öland, with the aim of understanding the relationships between sexual reproduction, habitat age, genetic variation and factors related to growth conditions. The overall proportion of fertile female shoots (with perichaetia) was 35%. Fertility status at the level of individual shoots was positively associated with shoot length and density, while within deme fertility was positively associated with bush cover. There was no association between female fertility and habitat age, genetic diversity (H_S) or allelic richness. Out of 1344 investigated shoots, only two were normal-sized fertile males. Dwarf males were also extremely rare, and found almost exclusively on shoots with sporophytes. Few sporophytes were observed (in the two demes with highest fertility and bush cover). No relationship between genetic variation and the frequency of sporophytes and males was found. The lack of a relationship between sexual reproduction and genetic variation suggests that sexual reproduction may not occur in the same grassland fragments as the recruitment of new clones (from spores or vegetative fragments). The majority of the dry, open grassland habitats, where H. lutescens is typically found in the study area, appear to be suboptimal for both dwarf males and fertilization. Sexual reproduction is more likely to occur in shaded (although grazed) grassland patches, where moisture levels are likely to be higher and the moss colonies are generally more vigorous.     

Isolation and biochemical characterization of underwater adhesives from diatoms

Many aquatic organisms are able to colonize surfaces through the secretion of underwater adhesives. Diatoms are unicellular algae that have the capability to colonize any natural and man-made submerged surfaces. There is great technological interest in both mimicking and preventing diatom adhesion, yet the biomolecules responsible have so far remained unidentified. A new method for the isolation of diatom adhesive material is described and its amino acid and carbohydrate composition determined. The adhesive materials from two model diatoms show differences in their amino acid and carbohydrate compositions, but also share characteristic features including a high content of uronic acids, the predominance of hydrophilic amino acid residues, and the presence of 3,4-dihydroxyproline, an extremely rare amino acid. Proteins containing dihydroxyphenylalanine, which mediate underwater adhesion of mussels, are absent. The data on the composition of diatom adhesives are consistent with an adhesion mechanism based on complex coacervation of polyelectrolyte-like biomolecules.     

Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.