Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids: substrate not required

Integration of thylakoid proteins by the chloroplast signal recognition particle (cpSRP) posttranslational transport pathway requires the cpSRP, an SRP receptor homologue (cpFtsY), and the membrane protein ALB3. Similarly, Escherichia coli uses an SRP and FtsY to cotranslationally target membrane proteins to the SecYEG translocase, which contains an ALB3 homologue, YidC. In neither system are the interactions between soluble and membrane components well understood. We show that complexes containing cpSRP, cpFtsY, and ALB3 can be precipitated using affinity tags on cpSRP or cpFtsY. Stabilization of this complex with GMP-PNP specifically blocks subsequent integration of substrate (light harvesting chl a/b-binding protein [LHCP]), indicating that the complex occupies functional ALB3 translocation sites. Surprisingly, neither substrate nor cpSRP43, a component of cpSRP, was necessary to form a complex with ALB3. Complexes also contained cpSecY, but its removal did not inhibit ALB3 function. Furthermore, antibody bound to ALB3 prevented ALB3 association with cpSRP and cpFtsY and inhibited LHCP integration suggesting that a complex containing cpSRP, cpFtsY, and ALB3 must form for proper LHCP integration.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

Microhabitat distribution of protostelids in a Tropical Wet Forest in Costa Rica

A microhabitat study of protostelids was carried out in a Tropical Wet Forest at the La Selva Biological Station in Costa Rica. Nine species were recorded from sterile wheat straws placed out and then re-collected over a period of six weeks from two different litter microhabitats in an area of primary forest. All nine species were present on straws placed in the aerial litter microhabitat, but only six species were present on straws placed in the forest floor litter microhabitat. Total colonies, percent of straws colonized, and mean number of species per straw increased significantly over time. One species (Schizoplasmodiopsis pseudoendospora) typical of temperate litter was the overwhelming dominant on the forest floor litter, while Echinostelium bisporum, a species rare in temperate litter microhabitats, was the single most abundant species in the aerial litter microhabitat. Both of these species had significantly increased frequencies over time. Two species abundant in temperate aerial litter microhabitats and one species abundant in temperate forest floor litter were rare at La Selva. Our data conform to those obtained in an earlier study carried out in tropical forests in the mountains of Puerto Rico and provide additional support towards developing a model of microhabitat distribution of protostelids in terrestrial ecosystems.     

Purification and preliminary characterization of brain aspartoacylase

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) in the brain to produce acetate and L-aspartate. An aspartoacylase deficiency, with concomitant accumulation of NAA, is responsible for Canavan disease, a lethal autosomal recessive disorder. To examine the mechanism of this enzyme the genes encoding murine and human aspartoacylase were cloned and expressed in Escherichia coli. A significant portion of the enzyme is expressed as soluble protein, with the remainder found as inclusion bodies. A convenient enzyme-coupled continuous spectrophotometric assay has been developed for measuring aspartoacylase activity. Kinetic parameters were determined with the human enzyme for NAA and for selected N-acyl analogs that demonstrate relaxed substrate specificity with regard to the nature of the acyl group. The clinically relevant E285A mutant reveals an altered enzyme with poor stability and barely detectable activity, while a more conservative E285D substitution leads to only fivefold lower activity than native aspartoacylase.     

ACTN3 genotype is associated with human elite athletic performance

There is increasing evidence for strong genetic influences on athletic performance and for an evolutionary "trade-off" between performance traits for speed and endurance activities. We have recently demonstrated that the skeletal-muscle actin-binding protein alpha-actinin-3 is absent in 18% of healthy white individuals because of homozygosity for a common stop-codon polymorphism in the ACTN3 gene, R577X. alpha-Actinin-3 is specifically expressed in fast-twitch myofibers responsible for generating force at high velocity. The absence of a disease phenotype secondary to alpha-actinin-3 deficiency is likely due to compensation by the homologous protein, alpha-actinin-2. However, the high degree of evolutionary conservation of ACTN3 suggests function(s) independent of ACTN2. Here, we demonstrate highly significant associations between ACTN3 genotype and athletic performance. Both male and female elite sprint athletes have significantly higher frequencies of the 577R allele than do controls. This suggests that the presence of alpha-actinin-3 has a beneficial effect on the function of skeletal muscle in generating forceful contractions at high velocity, and provides an evolutionary advantage because of increased sprint performance. There is also a genotype effect in female sprint and endurance athletes, with higher than expected numbers of 577RX heterozygotes among sprint athletes and lower than expected numbers among endurance athletes. The lack of a similar effect in males suggests that the ACTN3 genotype affects athletic performance differently in males and females. The differential effects in sprint and endurance athletes suggests that the R577X polymorphism may have been maintained in the human population by balancing natural selection.     

Reconciling actual and inferred population histories in the house finch (Carpodacus mexicanus) by AFLP analysis

The house finch (Carpodacus mexicanus) is a native songbird of western North America that was introduced to the eastern United States and Hawaiian Islands in historic times. As such, it provides an unusually good opportunity to test the ability of molecular markers to recover recent details of a known population history. To investigate this prospect, genetic variation in 172 individuals from 16 populations in the western and eastern United States, southeastern Canada, Hawaiian Islands, and Mexico, as well as genetic variation in the closely related purple finch (Carpodacus purpureus) and Cassin's finch (Carpodacus cassinii) was studied by a semi-automated fluorescence-labeled amplified fragment length polymorphism (AFLP) marker system. A total of 363 markers were generated, of which 258 (71.2%) were polymorphic among species, 166 (61.4%) polymorphic among house finch subspecies, and 157 (60.2%) polymorphic among populations within the frontalis subspecies complex. Heterozygosities and interpopulation divergences revealed by the analysis appeared relatively low at all taxonomic levels, but there are few similar studies in avian populations with which to compare results. Whereas the known population history predicts that both eastern and Hawaiian finches should have been derived from within western populations, tree analysis using both populations and individuals as units suggests weak monophyly of eastern populations and indicates that Hawaiian populations are not clearly derived from California populations. However, the genetic distinctiveness of native and recently founded populations was disclosed by analyses of molecular variance as well as by a model-based assignment approach in which 98%, 94%, and 99% individuals from western, Hawaiian, and eastern regions, respectively, were assigned correctly to their populations without using prior information on population of origin, suggesting that these recent introductions have resulted in detectable differentiation without substantial loss of AFLP diversity. Our results indicate that AFLPs are a useful tool for population genetic and evolutionary studies of birds, particularly as a prelude to finding molecular markers linked to traits subjected to recent adaptive evolution.     

OmpF enhances the ability of BtuB to protect susceptible Escherichia coli cells from colicin E9 cytotoxicity

The outer membrane (OM) vitamin B(12) receptor, BtuB, is the primary receptor for E group colicin adsorption to Escherichia coli. Cell death by this family of toxins requires the OM porin OmpF but its role remains elusive. We show that OmpF enhances the ability of purified BtuB to protect bacteria against the endonuclease colicin E9, demonstrating either that the two OM proteins form the functional receptor or that OmpF is recruited for subsequent translocation of the bacteriocin. While stable binary colicin E9-BtuB complexes could be readily shown in vitro, OmpF-containing complexes could not be detected, implying that OmpF association with the BtuB-colicin complex, while necessary, must be weak and/or transient in nature.     

Evidence that acute serotonergic activation potentiates the locomotor-stimulating effects of corticotropin-releasing hormone in juvenile chinook salmon (Oncorhynchus tshawytscha)

The present study investigated whether the serotonergic system is involved in mediating the behavioral effects of corticotropin-releasing hormone (CRH) in juvenile spring chinook salmon, Oncorhynchus tshawytscha. An intracerebroventricular (ICV) injection of CRH induced hyperactivity. The effect of CRH was potentiated in a dose-dependent manner by the concurrent administration of the serotonin (5-HT) selective reuptake inhibitor fluoxetine. However, administration of fluoxetine alone had no effect on locomotor activity, suggesting that the locomotor-stimulating effect of CRH is mediated by the activation of the serotonergic system. Conversely, ICV injections of the 5-HT(1A) receptor antagonist NAN-190 attenuated the effect of CRH on locomotor activity when given in combination with CRH but had no effect when administered alone. These results provide the first evidence to support the hypothesis that the effect of CRH on locomotor activity in teleosts is mediated by activating the serotonergic system.