Structure of pyrimidine 5'-nucleotidase type 1. Insight into mechanism of action and inhibition during lead poisoning

Eukaryotic pyrimidine 5'-nucleotidase type 1 (P5N-1) catalyzes dephosphorylation of pyrimidine 5'-mononucleotides. Deficiency of P5N-1 activity in red blood cells results in nonspherocytic hemolytic anemia. The enzyme deficiency is either familial or can be acquired through lead poisoning. We present the crystal structure of mouse P5N-1 refined to 2.35 A resolution. The mouse P5N-1 has a 92% sequence identity to its human counterpart. The structure revealed that P5N-1 adopts a fold similar to enzymes of the haloacid dehydrogenase superfamily. The active site of this enzyme is structurally highly similar to those of phosphoserine phosphatases. We propose a catalytic mechanism for P5N-1 that is also similar to that of phosphoserine phosphatases and provide experimental evidence for the mechanism in the form of structures of several reaction cycle states, including: 1) P5N-1 with bound Mg(II) at 2.25 A, 2) phosphoenzyme intermediate analog at 2.30 A, 3) product-transition complex analog at 2.35 A, and 4) product complex at 2.1A resolution with phosphate bound in the active site. Furthermore the structure of Pb(II)-inhibited P5N-1 (at 2.35 A) revealed that Pb(II) binds within the active site in a way that compromises function of the cationic cavity, which is required for the recognition and binding of the phosphate group of nucleotides.     

Distribution,phosphorylation, and activities of Hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection

The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.     

Submicronic Fungal Bioaerosols: High-Resolution Microscopic Characterization and Quantification

Submicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in mold-contaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min⁻¹ airflow. The overall median numbers of aerosolized submicronic particles were 2 × 10⁵ cm⁻², 2.6 × 10³ cm⁻², and 0.9 × 10³ cm⁻² for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.     

Discovery of 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent and orally bioavailable tissue-nonspecific alkaline phosphatase (TNAP) inhibitor

Tissue-nonspecific alkaline phosphatase (TNAP) is an ectoenzyme crucial for bone matrix mineralization via its ability to hydrolyze extracellular inorganic pyrophosphate (ePP_i), a potent mineralization inhibitor, to phosphate (P_i). By the controlled hydrolysis of ePP_i, TNAP maintains the correct ratio of P_i to ePP_i and therefore enables normal skeletal and dental calcification. In other areas of the body low ePP_i levels lead to the development of pathological soft-tissue calcification, which can progress to a number of disorders. TNAP inhibitors have been shown to prevent these processes via an increase of ePP_i. Herein we describe the use of a whole blood assay to optimize a previously described series of TNAP inhibitors resulting in 5-((5-chloro-2-methoxyphenyl)sulfonamido)nicotinamide (SBI-425), a potent, selective and oral bioavailable compound that robustly inhibits TNAP in vivo.     

Evolutionary Analysis of Mitogenomes from Parasitic and Free-Living Flatworms

Mitochondrial genomes (mitogenomes) are useful and relatively accessible sources of molecular data to explore and understand the evolutionary history and relationships of eukaryotic organisms across diverse taxonomic levels. The availability of complete mitogenomes from Platyhelminthes is limited; of the 40 or so published most are from parasitic flatworms (Neodermata). Here, we present the mitogenomes of two free-living flatworms (Tricladida): the complete genome of the freshwater species Crenobia alpina (Planariidae) and a nearly complete genome of the land planarian Obama sp. (Geoplanidae). Moreover, we have reanotated the published mitogenome of the species Dugesia japonica (Dugesiidae). This contribution almost doubles the total number of mtDNAs published for Tricladida, a species-rich group including model organisms and economically important invasive species. We took the opportunity to conduct comparative mitogenomic analyses between available free-living and selected parasitic flatworms in order to gain insights into the putative effect of life cycle on nucleotide composition through mutation and natural selection. Unexpectedly, we did not find any molecular hallmark of a selective relaxation in mitogenomes of parasitic flatworms; on the contrary, three out of the four studied free-living triclad mitogenomes exhibit higher A+T content and selective relaxation levels. Additionally, we provide new and valuable molecular data to develop markers for future phylogenetic studies on planariids and geoplanids.     

Structure Learning in Bayesian Sensorimotor Integration

Previous studies have shown that sensorimotor processing can often be described by Bayesian learning, in particular the integration of prior and feedback information depending on its degree of reliability. Here we test the hypothesis that the integration process itself can be tuned to the statistical structure of the environment. We exposed human participants to a reaching task in a three-dimensional virtual reality environment where we could displace the visual feedback of their hand position in a two dimensional plane. When introducing statistical structure between the two dimensions of the displacement, we found that over the course of several days participants adapted their feedback integration process in order to exploit this structure for performance improvement. In control experiments we found that this adaptation process critically depended on performance feedback and could not be induced by verbal instructions. Our results suggest that structural learning is an important meta-learning component of Bayesian sensorimotor integration.     

Size Dependent Male Reproductive Tactic in the Two-Spotted Goby (Gobiusculus flavescens)

Male investment in testes and sperm duct gland in the polygamous nest breeding two-spotted goby Gobiusculus flavescens (Fabricius) was investigated in relation to time in reproductive season and individual physical parameters. This small teleost fish is most likely the most abundant species found along the rocky shores of the North East Atlantic. The two-spotted goby has a single reproductive season, during which nest-caring males can raise several clutches of offspring. According to the literature the males are on average larger than the females. Here we report for the first time a population showing a reversal of this trend, with males on average being smaller than females, a difference likely caused by a large proportion of small males. Early in the breeding season these small males have typical sneaker characters, with relatively large testes and small seminal duct glands compared to the larger dominant territorial males. The presence of these two alternative male reproductive tactics is confirmed by histological studies, which shows the presence of sperm in the sperm duct glands (SDG) of smaller males, but not in the SDG of intermediate and larger males. To our knowledge, males with typical sneaker characters have not been reported in earlier studied populations of two-spotted goby. Interestingly we found that testes investment declined significantly over the course of the breeding season, and that this reduction was significantly more pronounced in small compared to the large males. Further, a significant increase in seminal duct gland (SDG) mass was observed for the smaller males over the breeding season. We propose that this indicates a possible shift in mating tactic by smaller males from a parasitic to a nest-holding tactic over the course of the breeding season. Thus, the observed size dependent plasticity in investment in SDG over time suggests that the reproductive tactic of G. flavescens is conditional, and possibly influenced by mate availability and male—male competition.     

Testing generalized allometries in allocation modeling within an individual-based simulation framework

The allocation of carbohydrates from photosynthesis to various plant compartments is a key process in ecophysiology and consequently an important element in process-based ecosystem modeling. In this study, we tested generalized empirical equations in a widely applied partitioning concept based on compartment-specific biomass allometries. For an 88-year chronosequence of European beech (Fagus sylvatica L.) in Austria, we used the individual-based hybrid forest model PICUS v1.4 to compare simulations employing foliage biomass functions at different levels of generalization against runs with site-specific parameterization and observations. Sensitivities of the individual tree model were generally in line with the original stand-level partitioning concept and ecological process understanding. While stand-level leaf area increased with increasing allocation to foliage, net primary productivity showed no significant response due to saturated radiation interception in the dense chronosequence stands. Strong sensitivities were revealed at the individual tree level, where favoring allocation to the foliage compartment resulted in increasing asymmetry of competition and height–diameter relationships. Applying a generalized parameterization based on data from the full range of continental species distribution resulted in a significant overestimation of mean tree height and subsequently standing volume stock at the chronosequence. At a lower hierarchical level of generality, however, simulations with a representative regional parameterization performed satisfactorily compared to model runs using the site-specific allometry. In relation to common accuracy demands, e.g., in forest management decision support, the study suggests the rejection of a generic parameterization while corroborating the use of regional generalizations in ecosystem models.