Phenoloxidase of peach (Prunus persica) endocarp: Its relationship with peroxidases and lignification

A preliminary characterization of a phenoloxidase from extracts of soluble and ionically-bound cell wall proteins of peach ( Prunus persica L. Batsch, cv. Redhaven) endocarp is described in the present study to establish differences with peroxidases from the same plant tissue. The phenoloxidase activity was detected mainly in the first stage of peach fruit growth, while peroxidase activity and lignin content increased along the second stage of growth. There were clear differences between the two enzymes. The phenoloxidase had a pI value of 5.6, different from those of peroxidases isoenzymes with various pIs ranging from 3.6 to 9.6. The oxidase molecular mass was 112 kDa, similar to other phenoloxidases described in the literature, while all peroxidase isoenzymes showed a molecular mass of around 40 kDa. The specific activities of phenoloxidase against different substrates and its inhibition by various effectors suggest that the endocarp oxidase described here is probably a metal-dependent polyphenol oxidase, displaying attributes of both catechol oxidase (EC 1.10.3.1) and laccase (EC 1.10.3.2).     

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and significant epigenetic modification. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in CpG context, allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. We used a CpG methyltransferase with a synthetic S-adenosyl-l-methionine cofactor analog to transfer an azide to cytosines instead of the natural methyl group. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased up to 2-fold by the addition of a nucleosidase, presumably by degrading the inactive by-product of the cofactor after labeling, preventing its inhibitory effect. We used the method to determine the decline in global DNA methylation in a chronic lymphocytic leukemia patient and then performed whole-genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published bisulfite sequencing methylation maps. Although mapping resolution is limited by optical detection to 500–1000 bp, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.     

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSF_M) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSF_M variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSF_M structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSF_M and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSF_M residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.     

Indole-3-acetic acid oxidase and syringaldazine oxidase activities of peroxidase isozymes in soybean root nodules

Many isoperoxidases with indole-3-acetic acid oxidase (IAA) and syringaldazine oxidase activities were detected by polyacrylamide gel electrophoresis in soybean root nodules [ Glycine max (L.) Merrill, cv. Asgrow], detached at the onset of flowering. The kinetics of the two activities were studied with some of the isoperoxidases partially purified by ion exchange chromatography. IAA oxidase activity of the cationic isoforms showed a sigmoidal kinetic behaviour and a higher substrate affinity than the anionic ones, whereas typical saturation kinetics were found with an anionic fraction that contained leghemoglobins. So, nodule IAA oxidase activity may mainly be displayed by the cationic isoforms. These cationic isoperoxidases had high affinity towards syringaldazine and they also may be associated with cell wall rigidification.     

Pollinator‐mediated selection on floral size and tube color in Linum pubescens: Can differential behavior and preference in different times of the day maintain dimorphism?

Diversity of flower traits is often proposed as the outcome of selection exerted by pollinators. Positive directional pollinator‐mediated selection on floral size has been widely shown to reduce phenotypic variance. However, the underlying mechanism of maintaining within‐population floral color polymorphism is poorly understood. Divergent selection, mediated by different pollinators or by both mutualists and antagonists, may create and maintain such polymorphism, but it has rarely been shown to result from differential behavior of one pollinator. We tested whether different behaviors of the same pollinators in morning and evening are associated with dimorphic floral trait in Linum pubescens, a Mediterranean annual plant that exhibits variable within‐population frequencies of dark‐ and light‐colored flower tubes. Usia bicolor bee‐flies, the major pollinators of L. pubescens, are mostly feeding in the flower in the morning, while in the evening they are mostly visiting the flowers for mating. In 2 years of studying L. pubescens in a single large population in the Carmel, Israel, we found in one year that dark‐centered flowers received significantly higher fraction of visits in the morning. Fitness was positively affected by number of visits, but no fitness differences were found between tube‐color morphs, suggesting that both morphs have similar pollination success. Using mediation analysis, we found that flower size was under positive directional pollinator‐mediated selection in both years, but pollinator behavior did not explain entirely this selection, which was possibly mediated also by other agents, such as florivores or a‐biotic stresses. While most pollinator‐mediated selection studies show that flower size signals food reward, in L. pubescens, it may also signal for mating place, which may drive positive selection. While flower size found to be under pollinator‐mediated selection in L. pubescens, differential behavior of the pollinators in morning and evening did not seem to explain flower color polymorphism.     

Role of the conserved carboxy-terminal α-helix of Rad6p in ubiquitination and DNA repair

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-terminal α-helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin-conjugating enzymes (Ubcs). This specific conservation suggests that the C-terminal a-helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error-free DNA repair in vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C-terminal u-helix is also essential for error-free DNA repair in vivo. Overexpression of Rad18p, a single-stranded DNA-binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C-terminal α-helix mutants to different degrees. This indicates that the C-terminal α-helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild-type capacity for error-free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.     

Late Quaternary reptile extinctions: size matters,insularity dominates

Aim A major Late Quaternary vertebrate extinction event affected mostly large‐bodied ‘megafauna’. This is well documented in both mammals and birds, but evidence of a similar trend in reptiles is scant. We assess the relationship between body size and Late Quaternary extinction in reptiles at the global level. Location Global. Methods We compile a body size database for all 82 reptile species that are known to have gone extinct during the last 50,000 years and compare them with the sizes of 10,090 extant reptile species (97% of known extant diversity). We assess the body size distributions in the major reptile groups: crocodiles, lizards, snakes and turtles, while testing and correcting for a size bias in the fossil record. We examine geographical biases in extinction by contrasting mainland and insular reptile assemblages, and testing for biases within regions and then globally by using geographically weighted models. Results Extinct reptiles were larger than extant ones, but there was considerable variation in extinction size biases among groups. Extinct lizards and turtles were large, extinct crocodiles were small and there was no trend in snakes. Lizard lineages vary in the way their extinction is related to size. Extinctions were particularly prevalent on islands, with 73 of the 82 extinct species being island endemics. Four others occurred in Australia. The fossil record is biased towards large‐bodied reptiles, but extinct lizards were larger than extant ones even after we account for this. Main conclusions Body size played a complex role in the extinction of Late Quaternary reptiles. Larger lizard and turtle species were clearly more affected by extinction mechanisms such as over exploitation and invasive species, resulting in a prevalence of large‐bodied species among extinct taxa. Insularity was by far the strongest correlate of recent reptile extinctions, suggesting that size‐biased extinction mechanisms are amplified in insular environments.     

Leishmania-sandfly interactions: an empirical field study

Phlebotomus papatasi is the sandfly vector of Leishmania major in the Jordan Valley. The objective of this study was to characterize vector-parasite relations in an active zoonotic focus. Seasonality and intensity of promastigote infection rates in female sandflies and the developmental stage of these hosts were established. On 153 trap-nights, 641 female P. papatasi were caught and examined. Of these, 48 (7.4%, range 12.9-4.8%) were infected with L. major promastigotes. Correlating the number of parasites with the gonotrophic age of the vector revealed that most infections initially are light ones, and those that survive in the vector generally prosper and proliferate. Comparing infection rates in parous and gravid flies revealed that similar proportions of gravid and parous flies are found infected. Thus, Leishmania infections do not appear to affect sandfly survival.     

Deletions in processed pseudogenes accumulate faster in rodents than in humans

Summary The relative rates of point nucleotide substitution and accumulation of gap events (deletions and insertions) were calculated for 22 human and 30 rodent processed pseudogenes. Deletion events not only outnumbered insertions (the ratio being 71 and 31 for human and rodent pseudogenes, respectively), but also the total length of deletions was greater than that of insertions. Compared with their functional homologs, human processed pseudogenes were found to be shorter by about 1.2%, and rodent pseudogenes by about 2.3%. DNA loss from processed pseudogenes through deletion is estimated to be at least seven times faster in rodents than in humans. In comparison with the rate of point substitutions, the abridgment of pseudogenes during evolutionary times is a slow process that probably does not retard the rate of growth of the genome due to the proliferation of processed pseudogenes.