Crystallographic evidence for dioxygen interactions with iron proteins

The interaction of dioxygen with iron plays a key role in many important biological processes, such as dioxygen transport in the bloodstream and the reduction of dioxygen by iron in respiration. However, the catalytic mechanisms employed, for example in ligand oxidation, are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. This review outlines the structural evidence obtained by X-ray crystallographic methods for the nature of the interactions between dioxygen and the metal in iron-containing proteins. Proteins involved in iron transport or electron transfer are not included.     

Phylogenetic reconstruction using secondary structures of Internal Transcribed Spacer 2 (ITS2, rDNA): finding the molecular and morphological gap in Caribbean gorgonian corals

Background  

Most phylogenetic studies using current methods have focused on primary DNA sequence information. However, RNA secondary structures are particularly useful in systematics because they include characteristics, not found in the primary sequence, that give "morphological" information. Despite the number of recent molecular studies on octocorals, there is no consensus opinion about a region that carries enough phylogenetic resolution to solve intrageneric or close species relationships. Moreover, intrageneric morphological information by itself does not always produce accurate phylogenies; intra-species comparisons can reveal greater differences than intra-generic ones. The search for new phylogenetic approaches, such as by RNA secondary structure analysis, is therefore a priority in octocoral research.     

Carry‐over Effects of Size at Metamorphosis in Red‐eyed Treefrogs: Higher Survival but Slower Growth of Larger Metamorphs

Most animals have complex life histories, composed of a series of ecologically distinct stages, and the transitions between stages are often plastic. Anurans are models for research on complex life cycles. Many species exhibit plastic timing of and size at metamorphosis, due to both environmental constraints on larval growth and development and adaptive plastic responses to environmental variation. Models predicting optimal timing of metamorphosis balance cost/benefit ratios across stages, assuming that size affects growth and mortality rates in each stage. Much research has documented such effects in the larval period, but we lack an equal understanding of juvenile growth and mortality. Here, we examine how variation in size at metamorphosis in the Neotropical red‐eyed treefrog, Agalychnis callidryas, affects post‐metamorphic growth, foraging, and behavior in the lab as well as growth and survival in the field. Surprisingly, many individuals lost mass for weeks after metamorphosis. In the lab, larger metamorphs lost more mass following metamorphosis, ate similar amounts, had lower food conversion efficiencies, and grew more slowly after mass loss ceased than did smaller ones. In field cages larger metamorphs were more likely to survive than smaller ones; just one froglet died in the lab. Our data suggest that size‐specific differences in physiology and behavior influence these trends. Comparing across species and studies, large size at metamorphosis generally confers higher survival; size effects on growth rates vary substantially among species, in both magnitude and direction, but may be stronger in the tropics.     

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55^Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4⁺ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P₂, therefore controlling Pr55^Gag membrane association. Rab27a also controls PI(4,5)P₂ levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55^Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P₂ production and, consequently, HIV-1 replication.     

Structural and functional properties of ryanodine receptor type 3 in zebrafish tail muscle

The ryanodine receptor (RyR)1 isoform of the sarcoplasmic reticulum (SR) Ca²⁺ release channel is an essential component of all skeletal muscle fibers. RyR1s are detectable as “junctional feet” (JF) in the gap between the SR and the plasmalemma or T-tubules, and they are required for excitation–contraction (EC) coupling and differentiation. A second isoform, RyR3, does not sustain EC coupling and differentiation in the absence of RyR1 and is expressed at highly variable levels. Anatomically, RyR3 expression correlates with the presence of parajunctional feet (PJF), which are located on the sides of the SR junctional cisternae in an arrangement found only in fibers expressing RyR3. In frog muscle fibers, the presence of RyR3 and PJF correlates with the occurrence of Ca²⁺ sparks, which are elementary SR Ca²⁺ release events of the EC coupling machinery. Here, we explored the structural and functional roles of RyR3 by injecting zebrafish (Danio rerio) one-cell stage embryos with a morpholino designed to specifically silence RyR3 expression. In zebrafish larvae at 72 h postfertilization, fast-twitch fibers from wild-type (WT) tail muscles had abundant PJF. Silencing resulted in a drop of the PJF/JF ratio, from 0.79 in WT fibers to 0.03 in the morphants. The frequency with which Ca²⁺ sparks were detected dropped correspondingly, from 0.083 to 0.001 sarcomere⁻¹ s⁻¹. The few Ca²⁺ sparks detected in morphant fibers were smaller in amplitude, duration, and spatial extent compared with those in WT fibers. Despite the almost complete disappearance of PJF and Ca²⁺ sparks in morphant fibers, these fibers looked structurally normal and the swimming behavior of the larvae was not affected. This paper provides important evidence that RyR3 is the main constituent of the PJF and is the main contributor to the SR Ca²⁺ flux underlying Ca²⁺ sparks detected in fully differentiated frog and fish fibers.     

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes

Klebsiella pneumoniae is an important cause of community‐acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3‐kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella‐containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV‐killed bacteria, the majority of live bacteria did not co‐localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K–Akt–Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down‐regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.     

Comparative Analysis of Genetic Chemotyping Methods for Fusarium: Tri13 Polymorphism Does not Discriminate between 3‐ and 15‐acetylated Deoxynivalenol Chemotypes in Fusarium graminearum

Genetic chemotyping is an essential tool for characterizing Fusarium populations causing head blight on wheat and other cereals. Three PCR methods, based on tri cluster polymorphism, were optimized and compared on 94 single‐spore isolates obtained from three continents belonging to F. gramineaurm, F. culmorum, F. poae, F. avenaceum and Microdochium nivale. While the methods based on the tri3, tri7 and tri12 polymorphism correctly identified all the tested strains, the method based on tri13 polymorphism was unable to discriminate between the 3‐ and 15‐acetylated DON forms in F. graminearum. It is advised to avoid the use of tri13 polymorphism for genetic chemotyping of the two acetylated chemotypes.     

Biodegradation of photo-degraded mulching films based on polyethylenes and stearates of calcium and iron as pro-oxidant additives

Polyethylene film materials persist in the environment for a long time. Several bacterial species have been isolated from films buried in soil located in Murcia, Spain. Bacterial strains were characterized with a combination of culture-dependent methods and sequencing of part of the 16S ribosomal RNA gene (rDNA) after amplification by polymerase chain reaction (PCR). Three bacterial species common in soil were found attached to the polymer and identified as Bacillus. cereus, B. megaterium, and B. subtilis. These microorganisms, as well as Brevibacillus borstelensis, were tested for biodegradation susceptibility at 30 and 45 °C on highly photo-degraded polyethylene films (500 h under irradiation of Xe-Lamp-solar filter) that contained calcium and iron stearates as pro-oxidant additives. Biofilm formation developed on the photo-degraded materials after one week of bacterial treatment. Biodegradation of the polyethylene films was studied by chemiluminescence, ATR-FTIR, and GC-product analysis and the data confirm a more efficient biodegradation on the bioassays carried out at higher temperature. The CL emissions due to decomposition of oxidation species take place at lower temperatures; the decrease of carbonyl index and the disappearance of photogenerated low-molecular products with biodegradation were more efficient on the biodegraded films at 45 °C. Also, mineralization was evaluated by carbon dioxide measurements using an indirect impedance technique. Biodegradation by B. borstelensis and MIX at 30 °C was slow and in the range of 0.7-1.2% of mineralization after 90 days of bacterial bioassay. At 45 °C biodegradation was more efficient and in particular in the more photo-degraded films containing Ca and Fe stearates where mineralization extents reached values of 11.5% with B. borstelensis and 7-10% with the mixture of Bacillus (MIX).