Monk parakeet invasion success: a role for nest thermoregulation and bactericidal potential of plant nest material?

Invasive species are a global threat to biodiversity, economy and human wellbeing. To mitigate these threats, identifying and halting the introduction of potentially invasive species is crucial. Although progress has been made in elucidating mechanisms underlying invasion success, the role of species behavioral strategies has only received scant attention. Here, we use the invasion of monk parakeets in Santa Catarina state, southern Brazil to study whether behavioral strategies such as nest thermoregulation and the ability to self-medicate against pathogens contribute to the establishment success of invading species. We relate data on monk parakeet reproductive success to ambient temperatures in- and outside nesting chambers and test the bactericidal potential of plants transported to the nest by breeding monk parakeets. Compared to breeding data from other invaded ranges and parts of the species’ native range, our results suggest both thermoregulation and the use of bactericidal plants could potentially influence monk parakeet reproductive success. Thermoregulation maintains stable temperatures of incubator chambers compared to large fluctuations (especially hotter extremes) outside the nest. At least one of the plants brought to the nest effectively inhibited growth of pathogenic bacteria. The union of these two factors could increase reproductive rates and may consequently aid the expansion of the species in new non-native environments.     

FT-IR difference spectroscopy elucidates crucial interactions of sensory rhodopsin I with the cognate transducer HtrI

The phototaxis receptor sensory rhodopsin I (SRI) from Halobacterium salinarum interacts with its cognate transducer (HtrI) forming a transmembrane complex. After light excitation of the chromophore all-trans retinal, SRI undergoes structural changes that are ultimately transmitted to HtrI. The interaction of SRI with HtrI results in the closure of the receptor's proton pathway, which renders the photocycle recovery kinetics of SRI pH-independent. We demonstrate on heterologously expressed and reconstituted SRI-HtrI fusion proteins that the transmembrane part of HtrI (residues 1-52) as well as the downstream cytoplasmic part (residues 53-147) exhibit conformational changes after light excitation. The sum of these conformational changes is similar to those observed in the fusion constructs SRI-HtrI 1-71 and SRI-HtrI 1-147, which display pH-independent receptor kinetics. These results indicate the occurrence of spatially distinct conformational changes that are required for functional signal transmission. Kinetic and spectroscopic analysis of HtrI point mutants of Asn53 provides evidence that this residue is involved in the receptor-transducer interaction. We suggest that Asn53 plays a role similar to that of Asn74 of the HtrII from Natronobacterium pharaonis, the latter forming a hydrogen bond to the receptor within the membrane.     

Temperature modulates binding specificity and affinity of the d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis

We investigated the effect of temperature on the binding specificity of the recombinant d-trehalose/d-maltose-binding protein from the hyperthermophilic archaeon Thermococcus litoralis (TMBP). Importantly, we found that TMBP can bind d-glucose (Glc). The Glc binding was characterized by means of fluorescence spectroscopy in the temperature range of 25 degrees C-85 degrees C. Our results show that at 25 degrees C the binding of Glc to TMBP is well represented by a bimodal model with apparent K(d) of 20 muM and approximately 3-8 mM for the first and the second binding step, respectively. At 60 degrees C the binding of Glc to TMBP is represented by a simple hyperbolic model with an apparent K(d) value of about 40 muM. Finally, at 85 degrees C Glc did not bind to TMBP. Molecular dynamics (MD) simulations were used to shed light on the molecular mechanism of the Glc binding. Our results suggest that after proper fluorescent labeling TMBP can be used as a highly thermostable and non-consuming analyte biosensor for monitoring the level of glucose in fluids (e.g. human blood) where other sugars are not present.     

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

Spatial and temporal Ca, Mg, and ATP dynamics in cardiac dyads during calcium release

We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP²⁻ with Ca²⁺ and Mg²⁺ ions on spatiotemporal concentration profiles of Ca²⁺, Mg²⁺, and ATP²⁻ in the dyadic cleft during Ca²⁺ release. The model revealed that Ca²⁺ concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca²⁺] in the dyadic space reached values similar to estimates of luminal [Ca²⁺] in ∼1 ms, suggesting that during calcium release the Ca²⁺ gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca²⁺ bound to ATP²⁻, and thus substantially decreased ATP²⁻ concentration in the dyadic space. The released Ca²⁺ could also replace Mg²⁺ in its complex with ATP²⁻ during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca²⁺, Mg²⁺, and ATP²⁻ might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.     

Unfolding kinetics of beta-lactoglobulin induced by surfactant and denaturant: a stopped-flow/fluorescence study

The beta-->alpha transition of beta-lactoglobulin, a globular protein abundant in the milk of several mammals, is investigated in this work. This transition, induced by the cationic surfactant dodecyltrimethylammonium chloride (DTAC), is accompanied by partial unfolding of the protein. In this work, unfolding of bovine beta-lactoglobulin in DTAC is compared with its unfolding induced by the chemical denaturant guanidine hydrochloride (GnHCl). The final protein states attained in the two media have quite different secondary structure: in DTAC the alpha-helical content increases, leading to the so-called alpha-state; in GnHCl the amount of ordered secondary-structure decreases, resulting in a random coil-rich final state (denatured, or D, state). To obtain information on both mechanistic routes, in DTAC and GnHCl, and to characterize intermediates, the kinetics of unfolding were investigated in the two media. Equilibrium and kinetic data show the partial accumulation of an on-pathway intermediate in each unfolding route: in DTAC, an intermediate (I(1)) with mostly native secondary structure but loose tertiary structure appears between the native (beta) and alpha-states; in GnHCl, another intermediate (I(2)) appears between states beta and D. Kinetic rate constants follow a linear Chevron-plot representation in GnHCl, but show a more complex mechanism in DTAC, which acts like a stronger binding species.     

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.     

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca²⁺ concentrations induce quasi-reversible diffusion of β1 integrins out of focal adhesions, whereas low Ca²⁺ concentrations induce irreversible recruitment of β1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated β1 receptors show that the cytoplasmic portion of β1 is required for low Ca²⁺-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified α-actinin colocalizes and redistributes with β1 receptors on ventral plasma membranes depleted of actin, implicating binding of α-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.     

Identification of a Novel Gene (ADPRTL1) Encoding a Potential Poly(ADP-ribosyl)transferase Protein

Poly(ADP-ribosyl)ation of nuclear proteins plays a significant role in the maintenance of genomic DNA stability. To date, four poly(ADP-ribosyl)ating proteins have been identified in humans. We now report the full-length sequence, expression profile, and chromosomal localization of a novel gene, ADPRTL1, encoding an ADP-ribosyltransferase-like protein. The predicted open reading frame encodes a protein of 1724 amino acids with a molecular mass of 192.8 kDa. The protein contains a region showing homology to the catalytic domains of the nuclear-localized ADP-ribosyltransferase proteins (Adprt), two recently identified Adprt-like proteins (Adprtl2 and Adprtl3), and the telomere-associated protein tankyrase. Key amino acids known to be important for the activity of these enzymes are conserved within this region of the Adprtl1 protein, indicating that Adprtl1 is a functional poly(ADP-ribosyl)transferase. As has been noted for tankyrase, sequence analysis of the Adprtl1 protein suggests that it is not capable of binding DNA directly. Thus, the transferase activity of Adprtl1 may be activated by other factors such as protein–protein interaction mediated by the extensive carboxyl terminus. We have subsequently refined the location of the ADPRTL1 genomic locus to 13q11, close to the recently cloned ZNF198 gene.