αB-Crystallin: A Hybrid Solid-State/Solution-State NMR Investigation Reveals Structural Aspects of the Heterogeneous Oligomer

Atomic-level structural information on αB-Crystallin (αB), a prominent member of the small heat-shock protein family, has been a challenge to obtain due its polydisperse oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on an ∼ 580-kDa human αB assembled from 175-residue 20-kDa subunits. An ∼ 100-residue α-crystallin domain is common to all small heat-shock proteins, and solution-state NMR was performed on two different α-crystallin domain constructs isolated from αB. In vitro, the chaperone-like activities of full-length αB and the isolated α-crystallin domain are identical. Chemical shifts of the backbone and C^β resonances have been obtained for residues 64-162 (α-crystallin domain plus part of the C-terminus) in αB and the isolated α-crystallin domain by solid-state and solution-state NMR, respectively. Both sets of data strongly predict six β-strands in the α-crystallin domain. A majority of residues in the α-crystallin domain have similar chemical shifts in both solid-state and solution-state, indicating similar structures for the domain in its isolated and oligomeric forms. Sites of intersubunit interaction are identified from chemical shift differences that cluster to specific regions of the α-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within αB. Evidence for a novel dimerization motif in the human α-crystallin domain is obtained by a comparison of (i) solid-state and solution-state chemical shift data and (ii) ¹H-¹⁵N heteronuclear single quantum coherence spectra as a function of pH. The isolated α-crystallin domain undergoes a dimer-monomer transition over the pH range 7.5-6.8. This steep pH-dependent switch may be important for αB to function optimally (e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia, which is accompanied by acidosis).     

Monitoring Insect Transposable Elements in Large Double-Stranded DNA Viruses Reveals Host-to-Virus and Virus-to-Virus Transposition

The mechanisms by which transposable elements (TEs) can be horizontally transferred between animals are unknown, but viruses are possible candidate vectors. Here, we surveyed the presence of host-derived TEs in viral genomes in 35 deep sequencing data sets produced from 11 host–virus systems, encompassing nine arthropod host species (five lepidopterans, two dipterans, and two crustaceans) and six different double-stranded (ds) DNA viruses (four baculoviruses and two iridoviruses). We found evidence of viral-borne TEs in 14 data sets, with frequencies of viral genomes carrying a TE ranging from 0.01% to 26.33% for baculoviruses and from 0.45% to 7.36% for iridoviruses. The analysis of viral populations separated by a single replication cycle revealed that viral-borne TEs originating from an initial host species can be retrieved after viral replication in another host species, sometimes at higher frequencies. Furthermore, we detected a strong increase in the number of integrations in a viral population for a TE absent from the hosts’ genomes, indicating that this TE has undergone intense transposition within the viral population. Finally, we provide evidence that many TEs found integrated in viral genomes (15/41) have been horizontally transferred in insects. Altogether, our results indicate that multiple large dsDNA viruses have the capacity to shuttle TEs in insects and they underline the potential of viruses to act as vectors of horizontal transfer of TEs. Furthermore, the finding that TEs can transpose between viral genomes of a viral species sets viruses as possible new niches in which TEs can persist and evolve.     

Sequence variability of Borna disease virus: resistance to superinfection may contribute to high genome stability in persistently infected cells

The RNA genome of Borna disease virus (BDV) shows extraordinary stability in persistently infected cell cultures. We performed bottleneck experiments in which virus populations from single infected cells were allowed to spread through cultures of uninfected cells and in which RNase protection assays were used to identify virus variants with mutations in a 535-nucleotide fragment of the M-G open reading frames. In one of the cell cultures, the major virus species (designated 2/1) was a variant with two point mutations in the G open reading frame. When fresh cells were infected with a low dose of a virus stock prepared from 2/1-containing cells, only a minority of the resulting persistently infected cultures contained detectable levels of the variant, whereas the others all seemed to contain wild-type virus. The BDV variant 2/1 remained stable in the various persistently infected cell cultures, indicating that the cells were resistant to superinfection by wild-type virus. Indeed, cells persistently infected with prototype BDV He/80 were also found to resist superinfection with strain V and vice versa. Our screen for mutations in the viral M and G genes of different rat-derived BDV virus stocks revealed that only one of four stocks believed to contain He/80 harbored virus with the original sequence. Two stocks mainly contained a novel virus variant with about 3% sequence divergence, whereas the fourth stock contained a mixture of both viruses. When the mixture was inoculated into the brains of newborn mice, the novel variant was preferentially amplified. These results provide evidence that the BDV genome is mutating more frequently than estimated from its invariant appearance in persistently infected cell cultures and that resistance to superinfection might strongly select against novel variants.     

The annual number of breeding adults and the effective population size of syntopic newts (Triturus cristatus, T. marmoratus)

Pond-breeding amphibians are deme-structured organisms with a population genetic structure particularly susceptible to demographic threats. We estimated the effective number of breeding adults (Nb) and the effective population size (Ne) of the European urodele amphibians Triturus cristatus (the crested newt) and T. marmoratus (the marbled newt), using temporal shifts in microsatellite allele frequencies. Eight microsatellite loci isolated from a T. cristatus library were used, five of which proved polymorphic in T. marmoratus, albeit with high frequencies of null alleles at two loci. Three ponds in western France were sampled, situated 4-10 kilometers apart and inhabited by both species. Parent-offspring cohort comparisons were used to measure Nb; samples collected at time intervals of nine or 12 years, respectively, were used to measure Ne. The adult population census size (N) was determined by mark-recapture techniques. With one exception, genetic distances (FST) between temporal samples were lower than among populations. Nb ranged between 10.6 and 101.8 individuals, Ne ranged between 9.6 and 13.4 individuals. For the pond where both parameters were available, Nb/N (overall range: 0.10-0.19) was marginally larger than Ne/N (overall range: 0.09-0.16), which is reflected in the temporal stability of N. In line with the observed differences in reproductive life-histories between the species, Nb/N ratios for newts were about one order of magnitude higher than for the anuran amphibian Bufo bufo. Despite of the colonization of the study area by T. cristatus only some decades ago, no significant genetic bottleneck could be detected. Our findings give rise to concerns about the long-term demographic viability of amphibian populations in situations typical for European landscapes.     

The Receptor-Like Protein ReMAX of Arabidopsis Detects the Microbe-Associated Molecular Pattern eMax from Xanthomonas

As part of their immune system, plants have pattern recognition receptors (PRRs) that can detect a broad range of microbe-associated molecular patterns (MAMPs). Here, we identified a PRR of Arabidopsis thaliana with specificity for the bacterial MAMP eMax from xanthomonads. Response to eMax seems to be restricted to the Brassicaceae family and also varied among different accessions of Arabidopsis. In crosses between sensitive accessions and the insensitive accession Shakhdara, eMax perception mapped to RECEPTOR-LIKE PROTEIN1 (RLP1). Functional complementation of rlp1 mutants required gene constructs that code for a longer version of RLP1 that we termed ReMAX (for receptor of eMax). ReMAX/RLP1 is a typical RLP with structural similarity to the tomato (Solanum lycopersicum) RLP Eix2, which detects fungal xylanase as a MAMP. Attempts to demonstrate receptor function by interfamily transfer of ReMAX to Nicotiana benthamiana were successful after using hybrid receptors with the C-terminal part of ReMAX replaced by that of Eix2. These results show that ReMAX determines specificity for eMax. They also demonstrate hybrid receptor technology as a promising tool to overcome problems that impede interfamily transfer of PRRs to enhance pathogen detection in crop plants.     

Strong male/male competition allows for nonchoosy females: high levels of polygynandry in a territorial frog with paternal care

Our knowledge about genetic mating systems and the underlying causes for and consequences of variation in reproductive success has substantially improved in recent years. When linked to longitudinal population studies, cross-generational pedigrees across wild populations can help answer a wide suite of questions in ecology and evolutionary biology. We used microsatellite markers and exhaustive sampling of two successive adult generations to obtain population-wide estimates of individual reproductive output of males and females in a natural population of the Neotropical frog Allobates femoralis (Aromobatidae), a pan-Amazonian species that features prolonged iteroparous breeding, male territoriality and male parental care. Parentage analysis revealed a polygynandrous mating system in which high proportions of males (35.5%) and females (56.0%) produced progeny that survived until adulthood. Despite contrasting reproductive strategies, successfully reproducing males and females had similar numbers of mating partners that sired the adult progeny (both sexes: median 2; range 1-6); the numbers of their offspring that reached adulthood were also similar (both sexes: median 2; range 1-8). Measures of reproductive skew indicate selection on males only for their opportunity to breed. Reproductive success was significantly higher in territorial than in nonterritorial males, but unrelated to territory size in males or to body size in both sexes. We hypothesize that female polyandry in this species has evolved because of enhanced offspring survival when paternal care is allocated to multiple partners.     

Short term protective effects of iron in a murine model of ischemia/reperfusion

The role of iron in the pathogenesis of cardio-vascular disorders is still controversial. We studied the effects of iron perturbations on myocardial injury upon temporary ischemia/reperfusion. C57BL/6J male mice were injected with iron dextran for 2 weeks while controls received saline. Mice were then subjected to 30 min of myocardial ischemia and subsequent reperfusion for 6–24 h. Tissue damage was quantified histologically and by troponin T determination. The expressions of tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) were investigated in non-ischemic and ischemic regions of both groups. After myocardial ischemia and reperfusion, troponin T levels, as a marker of myocardial damage, were significantly reduced in iron-treated mice as compared to control mice (P < 0.05). Under the same conditions the infarction area and damage score were significantly lower in iron-treated animals. In parallel, TNF-α and SOD expressions were increased in infarcted regions of iron-treated mice as compared to controls, whereas myocardial iNOS expression was significantly lower in iron-treated mice. Although, iron challenge increased radical formation and TNF-α expression in vivo, this did not result in myocardial damage which may be linked to the parallel induction of SOD. Importantly, iron treatment inhibited iNOS expression. Since, an increased nitric oxide (NO) formation has been linked to cardiac damage after acute myocardial infarction, iron may exert short time cardio-protective effects after induction of ischemia/reperfusion via decreasing iNOS formation. Both authors contributed equally to this work.