Ligand recombination to the alpha and beta subunits of human hemoglobin

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.     

Functional effects of heme orientational disorder in sperm whale myoglobin

The optical absorption and ligand binding properties of newly reconstituted sperm whale myoglobin were examined systematically at pH 8, 20 degrees C. The conventional absorbance and magnetic circular dichroism spectra of freshly reconstituted samples were identical to those of the native protein. In contrast, reconstituted azide or CO myoglobin initially exhibited less circular dichroism in the Soret wavelength region than native myoglobin. These data support the theory proposed by La Mar and co-workers (La Mar, G. N., Davis, N. L., Parish, D. W., and Smith, R. M. (1983) J. Mol. Biol. 168, 887-896) that protoheme inserts into apomyoglobin in two distinct orientations. The equilibrium and kinetic parameters for O2 and CO binding to newly reconstituted myoglobin were observed to be identical to those of the native protein. Thus, the orientation of the heme group has no effect on the physiological properties of myoglobin. This result is in disagreement with the preliminary report of Livingston et al. (Livingston, D. J., Davis, N. L., La Mar, G. N., and Brown, W. D. (1984) J. Am. Chem. Soc. 106, 3025-3026) which suggested that the abnormal heme conformation exhibited a 10-fold greater affinity and association rate constant for O2 binding. Significant kinetic heterogeneity was observed only for long-chain isonitrile binding to newly reconstituted myoglobin, and even in these cases, the rate constants for the abnormal and normal heme conformations differed by less than a factor of 4.     

Secondary chemicals protect mould from fungivory

The vast repertoire of toxic fungal secondary metabolites has long been assumed to have an evolved protective role against fungivory. It still remains elusive, however, whether fungi contain these compounds as an anti-predator adaptation. We demonstrate that loss of secondary metabolites in the soil mould Aspergillus nidulans causes, under the attack of the fungivorous springtail Folsomia candida, a disadvantage to the fungus. Springtails exhibited a distinct preference for feeding on a mutant deleted for LaeA, a global regulator of Aspergillus secondary metabolites. Consumption of the mutant yielded a reproductive advantage to the arthropod but detrimental effects on fungal biomass compared with a wild-type fungus capable of producing the entire arsenal of secondary metabolites. Our results demonstrate that fungal secondary metabolites shape food choice behaviour, can affect population dynamics of fungivores, and suggest that fungivores may provide a selective force favouring secondary metabolites synthesis in fungi.     

Survey of the fragile X syndrome CGG repeat and the short-tandem-repeat and single-nucleotide-polymorphism haplotypes in an African American population

Previous studies have shown that specific short-tandem-repeat (STR) and single-nucleotide-polymorphism (SNP)-based haplotypes within and among unaffected and fragile X white populations are found to be associated with specific CGG-repeat patterns. It has been hypothesized that these associations result from different mutational mechanisms, possibly influenced by the CGG structure and/or cis-acting factors. Alternatively, haplotype associations may result from the long mutational history of increasing instability. To understand the basis of the mutational process, we examined the CGG-repeat size, three flanking STR markers (DXS548-FRAXAC1-FRAXAC2), and one SNP (ATL1) spanning 150 kb around the CGG repeat in unaffected (n=637) and fragile X (n=63) African American populations and compared them with unaffected (n=721) and fragile X (n=102) white populations. Several important differences were found between the two ethnic groups. First, in contrast to that seen in the white population, no associations were observed among the African American intermediate or "predisposed" alleles (41-60 repeats). Second, two previously undescribed haplotypes accounted for the majority of the African American fragile X population. Third, a putative "protective" haplotype was not found among African Americans, whereas it was found among whites. Fourth, in contrast to that seen in whites, the SNP ATL1 was in linkage equilibrium among African Americans, and it did not add new information to the STR haplotypes. These data indicate that the STR- and SNP-based haplotype associations identified in whites probably reflect the mutational history of the expansion, rather than a mutational mechanism or pathway.     

Fruit,flies and filamentous fungi – experimental analysis of animal–microbe competition using Drosophila melanogaster and Aspergillus mould as a model system

In addition to their fundamental role in nutrient recycling, saprobiotic microorganisms may be considered as typical consumers of food‐limited ephemeral resource patches. As such, they may be engaged in inter‐specific competition with saprophagous animals feeding from the same resource. Bacteria and filamentous fungi are known to synthesise secondary metabolites, some of which are toxic and have been proposed to deter or harm animals. The microorganisms may, however, also be negatively affected if saprophagous animals do not avoid microbe‐laden resources but feed in the presence of microbial competitors. We hypothesised that filamentous fungi compete with saprophagous insects, whereby secondary metabolites provide a chemical shield against the insect competitors. For testing this, we developed a new ecological model system representing a case of animal–microbe competition between saprobiotic organisms, comprising Drosophila melanogaster and species of the fungus Aspergillus (A. nidulans, A. fumigatus, A. flavus). Infestation of Drosophila breeding substrate with proliferating fungal colonies caused graduated larval mortality that strongly depended on mould species and colony age. Confrontation with conidiospores only, did not result in significant changes in larval survival, suggesting that insect death may not be ascribed to pathogenic effects. When confronted with colonies of transgenic fungi that lack the ability to express the global secondary metabolite regulator LaeA (ΔlaeA), larval mortality was significantly reduced compared to the impact of the wild type strains. Yet, also in the ΔlaeA strains, inter‐specific variation in the influence on insect growth occurred. Competition with Drosophila larvae impaired fungal growth, however, wild type colonies of A. nidulans and A. flavus recovered more rapidly from insect competition than the corresponding ΔlaeA mutants (not in A. fumigatus). Our findings provide genetic evidence that toxic secondary metabolites synthesised by saprotrophic fungi may serve as a means to combat insect competitors. Variation in the ability of LaeA to control expression of various secondary metabolite gene clusters might explain the observed species‐specific variation in Drosophila–Aspergillus competition.     

Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice     

Clash of kingdoms or why Drosophila larvae positively respond to fungal competitors

BACKGROUND: Competition with filamentous fungi has been demonstrated to be an important cause of mortality for the vast group of insects that depend on ephemeral resources (e.g. fruit, dung, carrion). Recent data suggest that the well-known aggregation of Drosophila larvae across decaying fruit yields a competitive advantage over mould, by which the larvae achieve a higher survival probability in larger groups compared with smaller ones. Feeding and locomotor behaviour of larger larval groups is assumed to cause disruption of fungal hyphae, leading to suppression of fungal growth, which in turn improves the chances of larval survival to the adult stage. Given the relationship between larval density, mould suppression and larval survival, the present study has tested whether fungal-infected food patches elicit communal foraging behaviour on mould-infected sites by which larvae might hamper mould growth more efficiently. RESULTS: Based on laboratory experiments in which Drosophila larvae were offered the choice between fungal-infected and uninfected food patches, larvae significantly aggregated on patches containing young fungal colonies. Grouping behaviour was also visible when larvae were offered only fungal-infected or only uninfected patches; however, larval aggregation was less strong under these conditions than in a heterogeneous environment (infected and uninfected patches). CONCLUSION: Because filamentous fungi can be deadly competitors for insect larvae on ephemeral resources, social attraction of Drosophila larvae to fungal-infected sites leading to suppression of mould growth may reflect an adaptive behavioural response that increases insect larval fitness and can thus be discussed as an anti-competitor behaviour. These observations support the hypothesis that adverse environmental conditions operate in favour of social behaviour. In a search for the underlying mechanisms of communal behaviour in Drosophila, this study highlights the necessity of investigating the role of inter-kingdom competition as a potential driving force in the evolution of spatial behaviour in insects.     

Genetic Variation and the Role of Insect Life History Traits in the Ability of Drosophila Larvae to Develop in the Presence of a Competing Filamentous Fungus

Competitive interactions between organisms from distantly related phylogenetical branches have been suggested as being one of the most pervasive forms of interspecific competition. However, so-called inter-kingdom competition has rarely been the focus of ecological and evolutionary studies. Thus, a relatively novel hypothesis has been proposed on the basis that saprophagous insects might intensively compete with filamentous fungi for ephemeral resources (e.g. decaying plant tissue). Consideration that life history traits (e.g. developmental time) are adaptive in determining developmental success in the presence of con- or hetero-specifics competitors implies that these traits have been progressively established by natural selection. Because a similar scenario may apply to antagonistic interactions between saprophagous insects and filamentous fungi, one can expect the existence of heritable variation in developmental success when insect larvae are forced to grow in the presence of noxious mould. Therefore, this study aimed at discovering whether a local population of Drosophila melanogaster indeed harbours genetic variation in developmental success in the presence of the mould Aspergillus niger. By using the isofemale line technique, single larvae forced to feed on fungal infected or uninfected substrate were analysed for variation in survival probability to the adult stage, developmental time and body size of emerged adults. I found genetic variation in survival probability in fungal infected substrates but not in uninfected larval food sources. Mean developmental time and body size varied significantly among isofemale lines in both types of larval environment. Survival was negatively correlated with developmental time on fungal infected substrate, but variation in developmental time on fungal-free substrates was not correlated with survival on fungal infected food patches. Within-trait correlation between fungal infected and uninfected substrates was surprisingly weak, and developmental time was not correlated with body size. The results of this study demonstrate (a) the existence of genetic variation for larval developmental success in the presence of A. niger in a Drosophila population, and (b) heritability of important insect life history traits differed as a function of the larval environment (fungal infected or uninfected feeding substrate). I discuss models that might explain heritability differences and the evolutionary consequences of these results.     

Host–parasitoid interaction as affected by interkingdom competition

Although still underrepresented in ecological research, competitive interactions between distantly related organisms (so-called “interkingdom competition”) are expected to be widespread in various ecosystems, with yet unknown consequences for, e.g. trophic interactions. In the model host–parasitoid system Drosophila melanogaster–Asobara tabida, toxic filamentous fungi have been shown to be serious competitors that critically affect the density-dependent survival of host Drosophila larvae. This study investigates the extent to which the competing mould Aspergillus niger affects key properties of the well-studied Drosophila–parasitoid system and how the host–parasitoid interaction influences the microbial competitor. In contrast to slightly positive density-dependent host mortality under mould-free conditions, competing A. niger mediated a strong Allee effect for parasitised larvae, i.e. mortality decreased with increasing larval density. It was found that the common toxic fungal metabolite kojic acid is not responsible for higher death rates in parasitised larvae. Single parasitised Drosophila larvae were less harmful to fungal reproduction than unparasitised larvae, but this effect vanished with an increase in larval density. As predicted from the negative effect of fungi on host survival and thus on parasitoid fitness at low larval densities, A. tabida females spent less time foraging in fungus-infested patches. Interestingly, even though high host larval densities increased host survival, parasitoids still reduced their search efforts in fungus-infested patches, indicating a benefit for host larvae from feeding in the presence of noxious mould. Thus, this experimental study provides evidence of the potentially important role of interkingdom competition in determining trophic interactions in saprophagous animal communities and the dynamics of both host–parasitoid and microbial populations.     

Competition with filamentous fungi and its implication for a gregarious lifestyle in insects living on ephemeral resources

Abstract. 1. Recent studies have demonstrated the existence of positive density dependence in the survival and development of Drosophila (the so‐called Allee effect); however the underlying mechanisms of such Allee effects have remained elusive. Competition with filamentous fungi have often been suggested to be involved in causing high mortality at low larval density, but it has not yet been explicitly tested if the well known spatial aggregation of insect eggs yields a fitness benefit for the developing larvae in the presence of noxious moulds. 2. Using Drosophila melanogaster, the present study tested whether larval survival is greater in aggregations when confronted with various combinations of three representative mould species (Aspergillus, Alternaria, and Penicillium) and a head start for fungal development. 3. High rates of fungal‐dependent mortality and significant positive density‐dependent larval survival (i.e. Allee effects) were observed when larvae were confronted with food resources containing established colonies of Aspergillus or Alternaria. Neither the simultaneous transfer of Aspergillus or Alternaria spores with larvae to food patches nor food infections with Penicillium affected insect larval development. 4. Significant correlations between mould growth and larval survival could be identified, although the patterns that emerged were shown to be inconsistent when the effects were compared between fungal species and fungal priority. Because mould growth only partly explained larval survival, the influence of other fungal‐borne factors, e.g. mycotoxins, needs to be elucidated in order to understand the mechanistic basis of insect–mould interactions. 5. These results are the first to argue convincingly for moulds being involved in mediating Allee effects for insects on ephemeral resources; however they also demonstrate an unexpected diversity in insect–mould interactions. Considering this diversity may be important in understanding insect spatial ecology.