Reductase domain of Drosophila melanogaster nitric-oxide synthase: redox transformations, regulation, and similarity to mammalian homologues

The nitric oxide synthase of Drosophila melanogaster (dNOS) participates in essential developmental and behavioral aspects of the fruit fly, but little is known about dNOS catalysis and regulation. To address this, we expressed a construct comprising the dNOS reductase domain and its adjacent calmodulin (CaM) binding site (dNOSr) and characterized the protein regarding its catalytic, kinetic, and regulatory properties. The Ca2+ concentration required for CaM binding to dNOSr was between that of the mammalian endothelial and neuronal NOS enzymes. CaM binding caused the cytochrome c reductase activity of dNOSr to increase 4 times and achieve an activity comparable to that of mammalian neuronal NOS. This change was associated with decreased shielding of the FMN cofactor from solvent and an increase in the rate of NADPH-dependent flavin reduction. Flavin reduction in dNOSr was relatively slow following the initial 2-electron reduction, suggesting a slow inter-flavin electron transfer, and no charge-transfer complex was observed between bound NADP+ and reduced FAD during the process. We conclude that dNOSr catalysis and regulation is most similar to the mammalian neuronal NOS reductase domain, although differences exist in their flavin reduction behaviors. The apparent conservation between the fruit fly and mammalian enzymes is consistent with dNOS operating in various signal cascades that involve NO.     

Spiroplasma species in the Costa Rican highlands: implications for biogeography and biodiversity

More than 1,000 Spiroplasma isolates have been obtained from horse flies and deer flies (Diptera:Tabanidae) in the United States and Canada. However, the spiroplasma biota of Central America is poorly known. In August of 1995 and 1998, 13 isolates were obtained in 14 attempts from horse flies of a single species, Poeciloderas quadripunctatus, taken in the Costa Rican highlands (1,100–2,000 m). The majority of the “isolates” proved to be mixtures of two or more Spiroplasma species, but after filter cloning, single strains emerged that were designated as representatives of the 13 accessions. Six distinct spiroplasma serogroups were identified from these isolations. Three of the strains are putative new species with no serological relationship to any other Spiroplasma species. A fourth strain is a putative new species that may be distantly related to S. helicoides, a southeastern U.S. species. These four strains are accorded herein status as representatives of new serogroups: strain BARC 4886 (group XXXV); strain BARC 4900 (group XXXVI); strain BARC 4908 (group XXXVII); and GSU5450 (group XXXVIII). A fifth Spiroplasma species was very closely related to S. lineolae, known previously only from the Georgia (U.S.) coast. The sixth was most closely related to subgroup VIII-3, known from Texas and the southeastern U.S. Discovery of six spiroplasma species in only 13 attempted isolations reflects diversity seldom equaled in southeast Georgia, and never elsewhere in the U.S. These results are consistent with a hypothesis that spiroplasma diversity increases from north (Nova Scotia) to south (Georgia and Costa Rica). The discovery of significant affinity between some spiroplasmas of the southeastern U.S. and the Costa Rican highlands was unexpected, but may reflect a climatically complex Pleistocene history.

Improvement of sordarin production through process optimization: combining traditional approaches with DOE

BMS-353645, also known as sordarin, was of interest based on its activity against pathogenic fungi. The objective of these studies was to provide high quality starting substrate for chemical modification aimed at further improving biological activity, with particular interest in the inhibition of Aspergillus. In the work presented here, Design of Experiments, or DOE, was successfully combined with traditional approaches to significantly improve sordarin yields in fermentation flasks. Overall, yields were increased 25-fold from <100 μg/g to as high as 2,609 μg/g in flasks through the use of various medium and conduction changes supplemented with DOE. The improved process was then successfully scaled to pilot plant tanks with the best batch producing 2,389 μg/g sordarin at the 250-l scale.     

Keeping in check painful synapses in central amygdala

Glutamatergic projections from the parabrachial nucleus to the central amygdala are implicated in pain transmission. In this issue of Neuron, Delaney et al. identify a new form of adrenergic modulation at these synapses, demonstrating that noradrenaline-induced suppression of glutamate release is mediated by a decrease in the number of sites of synaptic transmission without changes in probability of release.     

Design and synthesis of arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 1

A series of Nalpha-acyl-alpha-amino acid-(arylaminoethyl)amides were found to be potent and noncovalent cathepsin S inhibitors. Compound 20 possessed high cathepsin S affinity (Ki=3.3 nM) and showed excellent selectivity over cathepsin K, L, F, and V. Molecular modeling, design, synthesis, and in vitro activity are described.     

A Standard Reference Material to determine the sensitivity of techniques for detecting low-frequency mutations, SNPs, and heteroplasmies in mitochondrial DNA

Human mitochondrial DNA (mtDNA) mutations are important for forensic identifications and mitochondrial disease diagnostics. Low-frequency mutations, heteroplasmies, or SNPs scattered throughout the DNA in the presence of a majority of mtDNA with the Cambridge Reference Sequence (CRS) are almost impossible to detect. Therefore, the National Institute of Science and Technology has developed heteroplasmic human mtDNA Standard Reference Material (SRM) 2394 to allow scientists to determine their sensitivity in detecting such differences. SRM 2394 is composed of mixtures ranging from 1/99 to 50/50 of two 285-bp PCR products from two cell lines that differ at one nucleotide position. Twelve laboratories using various mutation detection methods participated in a blind interlaboratory evaluation of a prototype of SRM 2394. Most of these procedures were unable to detect the mutation when present below 20%, an indication that, in many real-life cases, low-frequency mutations remain undetected and that more sensitive mutation detection techniques are urgently needed.     

Untangling a Decline in Tropical Forest Resilience: Constraints on the Sustainability of Shifting Cultivation Across the Globe

Shifting cultivators depend on forest biomass inputs to nourish their crops. For them, forest resilience has an immediate impact: it affects crop productivity. A decline in the rate of recovery following shifting cultivation would ultimately affect local, regional and global carbon budgets, with feedbacks to climate. Yet the long-term impacts of shifting cultivation have been quantified in only six locations. In this study, we reanalyze data from these locations to determine whether the rate of biomass recovery is the same from cycle to cycle. Further, using case studies in Southern Yucatan, Mexico and West Kalimantan, Indonesia, we investigate the ecological and socioeconomic factors that affect forest resilience and thus determine whether or not shifting cultivation is sustainable. The reanalysis links aboveground biomass recovery following shifting cultivation to site productivity, forest age, fallow length, history of cultivation, and soil texture. Across locations, biomass accumulation rate declines by 9.3 percent with each cycle of shifting cultivation. Per cycle change in biomass accumulation rate is significantly more negative in younger forests and forests that experience a shorter fallow period. However, more detailed analyses for two case studies suggest that a purely ecological framework is of limited effectiveness in explaining variability in the effect of repeated shifting cultivation. Rather, socioeconomic factors such as migration, subsidies, roads, and settlement history can alter the outcome of shifting cultivation by limiting the accumulation and use of local knowledge.     

Reproductive Flexibility: Genetic Variation,Genetic Costs and Long-Term Evolution in a Collembola

In a variable yet predictable world, organisms may use environmental cues to make adaptive adjustments to their phenotype. Such phenotypic flexibility is expected commonly to evolve in life history traits, which are closely tied to Darwinian fitness. Yet adaptive life history flexibility remains poorly documented. Here we introduce the collembolan Folsomia candida, a soil-dweller, parthenogenetic (all-female) microarthropod, as a model organism to study the phenotypic expression, genetic variation, fitness consequences and long-term evolution of life history flexibility. We demonstrate that collembola have a remarkable adaptive ability for adjusting their reproductive phenotype: when transferred from harsh to good conditions (in terms of food ration and crowding), a mother can fine-tune the number and the size of her eggs from one clutch to the next. The comparative analysis of eleven clonal populations of worldwide origins reveals (i) genetic variation in mean egg size under both good and bad conditions; (ii) no genetic variation in egg size flexibility, consistent with convergent evolution to a common physiological limit; (iii) genetic variation of both mean reproductive investment and reproductive investment flexibility, associated with a reversal of the genetic correlation between egg size and clutch size between environmental conditions ; (iv) a negative genetic correlation between reproductive investment flexibility and adult lifespan. Phylogenetic reconstruction shows that two life history strategies, called HIFLEX and LOFLEX, evolved early in evolutionary history. HIFLEX includes six of our 11 clones, and is characterized by large mean egg size and reproductive investment, high reproductive investment flexibility, and low adult survival. LOFLEX (the other five clones) has small mean egg size and low reproductive investment, low reproductive investment flexibility, and high adult survival. The divergence of HIFLEX and LOFLEX could represent different adaptations to environments differing in mean quality and variability, or indicate that a genetic polymorphism of reproductive investment reaction norms has evolved under a physiological tradeoff between reproductive investment flexibility and adult lifespan.     

Regional distribution of blood flow within the diaphragm

We investigated the regional distribution of blood flow (Q) within the costal and crural portions of the diaphragm in a total of eight anesthetized supine mongrel dogs. Q was measured with 15-microns microspheres, radiolabeled with three different isotopes, injected into the left ventricle during spontaneous breathing (SB), inspiratory resistive loading (IR), and mechanical ventilation after paralysis (P). At necropsy, the costal and crural portions of each hemidiaphragm were arbitrarily subdivided along a sagittal plane into five to seven and three sections, respectively. During P, there was a dorsoventral Q gradient within the costal part of the diaphragm. During SB there was a fourfold increase in the gradient of Q. Furthermore, during IR, in which mouth pressures of -16 +/- 4 cmH2O were generated, there was a further increase in the gradient of Q. During both SB and IR, Q to the most ventral portion of the costal diaphragm was 26 +/- 6% less than the peak value. In two dogs, studied prone and supine, there was no difference in the Q gradients between the two postures. Over the dorsal 80% of the costal diaphragm there was also a dorsoventral gradient of muscle thickness, such that the most dorsal part was 54 +/- 2% (n = 5) that of the ventral portion. In contrast, there was no consistent gradient of Q or muscle thickness within the crural diaphragm. Our results demonstrate a topographical gravity-independent distribution of Q in the costal, but not the crural, diaphragm.(ABSTRACT TRUNCATED AT 250 WORDS)