Redefining dominance calculation: Increased competition flattens the dominance hierarchy in dairy cows.

Dominance hierarchies are known for mitigating conflicts and guiding priority of access to limited resources in gregarious animals. The dominance hierarchy of dairy cows is typically investigated using agonistic interactions, often monitored at the feed bunk right after fresh feed delivery when competition is high resulting in frequent interactions. Yet, the outcome of agonistic interactions during times of high competition may be more influenced by cows' high valuation of fresh feed than their intrinsic attributes, such that the dominance hierarchy constructed using agonistic interactions under high versus low competition times might differ. We tested how the structure of the dominance hierarchy changes in relation to different levels of competition in a dynamic group of 48 lactating dairy cows over 10 mo, with 6 cows exchanged every 16 d for a total of 159 cows. Using a validated algorithm we continuously detected the actor and reactor of replacement behaviors in 30 feed bins as cows competed for feed. We also calculated the percentage of occupied feed bins to characterize competition at the moment of each replacement. These data were combined to create hierarchies using Elo ratings, separately for 25 occupancy levels ranging from 13% to 100%. For each 1% rise in feeder occupancy, hierarchy steepness fell by 2.41 × 10-3 ± 9.71 × 10-5 (SE), and the percentage of dyads where both cows replaced each other rose by 0.13% ± 0.01%. At the highest feeder occupancy level in comparison to the lowest one, we observed 7.57% more dyads in which the dominant individual (those won more interactions at the lowest feeder occupancy) started to lose proportionally more. The magnitude of decrease in the winning rate of the dominant individual in those dyads also got amplified by 1.06 × 10-3% ± 1.37 × 10-4% (SE) for each 1% increase in feeder occupancy. These findings illustrate how inferred hierarchies vary with competition, with high competition flattening the hierarchy due to increased success of subordinate animals. We suggest that during heightened competition, increased valuation of resources can affect competitive success more than the individual's intrinsic dominance attributes. We recommend against calculating dominance hierarchies based on agonistic interactions during periods of high competition alone, and more generally urge researchers to differentiate agonistic interactions based on context when constructing dominance hierarchies.

Effects of group size on agonistic interactions in dairy cows: a descriptive study.

Group-housed cattle may engage in agonistic interactions over resources such as feed, which can negatively affect aspects of welfare. Little is known about how contextual factors such as group size influence agonistic behaviour. We explored the frequency of agonistic interactions at the feeder when cattle were housed in different-sized groups. We also explored the consistency of the directionality of agonistic interactions in dyads and of the number of agonistic interactions initiated by individuals across the group sizes. Four replicates of 50 cows each were assessed in two group-size phases. In Phase 1, cows were kept in one group of 50. In Phase 2, these same cows were divided into five groups of 10, maintaining stocking density (i.e., ratio of animals to lying stalls and feed bunk spaces). We measured agonistic replacements (i.e., interactions that result in one cow leaving the feed bin and another taking her place) at an electronic feeder using a validated algorithm. We used these data from Phase 1 to calculate individual Elo-ratings (a type of dominance score). Cows were then categorised into five dominance categories based upon these ratings. To ensure a consistent Elo-rating distribution between phases, two cows from each dominance category were randomly assigned to each small group of 10 cows. The mean ± SE number of replacements per cow was similar regardless of whether the cows were housed in groups of 50 (34.1 ± 2.4) or 10 (31.1 ± 4.5), although the groups of 10 were more variable. Further, 81.6 ± 7.7% (mean ± SD) of dyads had the same directionality across group sizes (i.e., the same individual won the majority of interactions in the dyad) and individuals were moderately consistent in the number of replacements they initiated (intraclass correlation coefficient = 0.62 ± 0.11; mean ± SD). These results indicate that the relationship between group size and agonistic behaviour is complex; we discuss these challenges and suggest new avenues for further research.

The Effect of Placement and Group Size on the Use of an Automated Brush by Groups of Lactating Dairy Cattle.

Mechanical brushes are often provided on dairy farms to facilitate grooming. However, current brush designs do not provide data on their use, and thus little is known about the effects of group size and placement of brushes within the pen. The objectives of this study were to automatically detect brush use in cow groups and to investigate the influence of (1) group size and the corresponding cow-to-brush ratio and (2) brush placement in relation to the lying stalls and the feeding and drinking areas. We measured brush use in groups of 60, 48, 36, and 24 cows, with the brush placed either in the alley adjacent to the feed bunk and water trough or in the back alley. Cows used the brush for longer when it was placed in the feed/water alley compared to when placed in the back alley. Average brush use per cow increased when cows were housed in smaller groups, but the brush was never in use more than 50% of the day, regardless of group size. We conclude that brush use increases when availability is increased and when the brush is placed closer to the feed and water.

Invited review: Social dominance in dairy cattle: A critical review with guidelines for future research.

Cattle are gregarious animals able to form social relationships. Dominance is one of the most widely studied social behaviors of dairy cattle, especially cows confined indoors. However, much of the past dairy cattle research has used an unstandardized approach, differing in definitions and conceptual understanding of dominance, as well as their methods of data collection and dominance calculation. The first of the 3 aims of this review is to evaluate how dominance relates to the social behavior of housed dairy cows. Cows engage in agonistic interactions to establish and reinforce dominance relationships. An individual's dominance is influenced by intrinsic characteristics, such as personality, and extrinsic factors, including group composition. When competing for resources, agonistic interactions can also be influenced by individual motivational differences, such as hunger, which may diminish the role of dominance in regulating competition. Our second aim is to critically review methods used to assess dominance in cows. This includes discussions on the effect of time and location of data collection on measured values as well as the viability and limitations of some dominance calculation methods. We propose that different methodologies lend themselves to different types of research questions. For example, the use of data stream-based methods that consider the sequence of interactions are useful for estimating how dominance fluctuates with changing conditions and can be used in a dynamically changing group. In contrast, matrix-based methods that aggregate social interactions may be best for identifying the social position of individuals and understanding how social characteristics influence the attributes of a stable hierarchy. Our third aim is to discuss the future of dominance research. We use a flowchart to illustrate guidelines for a more standardized approach to measuring dominance in cattle. We also identify areas in need of further conceptual clarification, suggest practical applications of dominance when managing dairy cattle, and discuss some limitations of dominance research.

The effects of cow dominance on the use of a mechanical brush.

An animal's social position within a group can influence its ability to perform important behaviours like eating and resting, but little is known about how social position affects the ability to express what are arguably less important but still rewarding behaviors, such as grooming. We set out to assess if dominance measured at the feeder is associated with increased use of a mechanical brush. Over a 2-year period, 161 dry cows were enrolled in a dynamically changing group of 20 individuals with access to a mechanical brush. We determined dominance using agonistic behaviors at the feeder and retrospectively analyzed brush use for the 12 most, and 12 least dominant individuals during the week before calving. Cows that were more dominant at the feeder used the brush more, especially during peak feeding times. Agonistic interactions at the brush did not differ between dominants and subordinates and were not related to brushing duration. These findings indicate that social position, calculated using competition for feed, affects mechanical brush access such that subordinates use the brush less than dominant cows independent of competition or time of day.

Structure of human ADAM-8 catalytic domain complexed with batimastat.

The role of ADAM-8 in cancer and inflammatory diseases such as allergy, arthritis and asthma makes it an attractive target for drug development. Therefore, the catalytic domain of human ADAM-8 was expressed, purified and crystallized in complex with a hydroxamic acid inhibitor, batimastat. The crystal structure of the enzyme-inhibitor complex was refined to 2.1 Å resolution. ADAM-8 has an overall fold similar to those of other ADAM members, including a central five-stranded ß-sheet and a catalytic Zn(2+) ion. However, unique differences within the S1' binding loop of ADAM-8 are observed which might be exploited to confer specificity and selectivity to ADAM-8 competitive inhibitors for the treatment of diseases involving this enzyme.

Dissecting the substrate recognition of 3-O-sulfotransferase for the biosynthesis of anticoagulant heparin.

Heparin is a polysaccharide-based natural product that is used clinically as an anticoagulant drug. Heparan sulfate 3-O-sulfotransferase (3-OST) is an enzyme that transfers a sulfo group to the 3-OH position of a glucosamine unit. 3-OST is present in multiple isoforms, and the polysaccharides modified by these different isoforms perform distinct biological functions. 3-OST isoform 1 (3-OST-1) is the key enzyme for the biosynthesis of anticoagulant heparin. Here, we report the crystal structure of the ternary complex of 3-OST-1, 3'-phosphoadenosine 5'-phosphate, and a heptasaccharide substrate. Comparisons to previously determined structures of 3-OST-3 reveal unique binding modes used by the different isoforms of 3-OST for distinguishing the fine structures of saccharide substrates. Our data demonstrate that the saccharide substrates display distinct conformations when interacting with the different 3-OST isoforms. Site-directed mutagenesis data suggest that several key amino residues, including Lys259, Thr256, and Trp283 in 3-OST-3 and Arg268 in 3-OST-1, play important roles in substrate binding and specificity between isoforms. These results deepen our understanding of the biosynthetic mechanism of heparan sulfate and provide structural information for engineering enzymes for an enhanced biosynthetic approach to heparin production.

The structural basis for partitioning of the XRCC1/DNA ligase III-α BRCT-mediated dimer complexes.

The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-α. For efficient ligation, ligase III-α is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-α BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex.

Conformational dependence of 13C shielding and coupling constants for methionine methyl groups.

Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of (3)J (CSCC), (3)J (CSCH), and the isotropic shielding, sigma(iso). Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-(13)C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, (3)J (CSCC)/(3)J (COCC) approximately 0.7. It is further demonstrated that the (3)J (CSCH) coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of delta(13)Cepsilon. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large approximately 100 degrees chi(3) values. The utility of the delta(13)Cepsilon as a basis for estimating the gauche/trans ratio for chi(3) is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.

Der p 5 crystal structure provides insight into the group 5 dust mite allergens.

Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of approximately 3000 A(3) that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

A 5' cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs.

A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the "core" PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

Template strand scrunching during DNA gap repair synthesis by human polymerase lambda.

Family X polymerases such as DNA polymerase lambda (Pol lambda) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5' and 3' ends of short gaps. DNA binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol lambda scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ when 2-nt gaps are involved. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair.

Slow conformational dynamics at C2'-endo nucleotides in RNA.

RNA molecules undergo local conformational dynamics on timescales spanning picoseconds to minutes. Slower local motions have the greater potential to govern RNA folding, ligand recognition, and ribonucleoprotein assembly reactions but are difficult to detect in large RNAs with complex structures. RNA SHAPE chemistry employs acylation of the ribose 2'-hydroxyl position to measure local nucleotide flexibility in RNA and is well-characterized by a mechanism in which each nucleotide samples unreactive (closed) and reactive (open) states. We monitor RNA conformational dynamics over distinct time domains by varying the electrophilicity of the acylating reagent. Select C2'-endo nucleotides are nonreactive toward fast reagents but reactive toward slower SHAPE reagents in both model RNAs and in a large RNA with a tertiary fold. We conclude, first, that the C2'-endo conformation by itself does not govern SHAPE reactivity. However, some C2'-endo nucleotides undergo extraordinarily slow conformational changes, on the order of 10(-4) s(-1). Due to their distinctive local dynamics, C2'-endo nucleotides have the potential to function as rate-determining molecular switches and are likely to play central, currently unexplored, roles in RNA folding and function.

Crystal structure of a type II dihydrofolate reductase catalytic ternary complex.

Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms a symmetric homotetramer with a central pore which functions as the active site. Its unusual structure, which results in a promiscuous binding surface that accommodates either the dihydrofolate (DHF) substrate or the NADPH cofactor, has constituted a significant limitation to efforts to understand its substrate specificity and reaction mechanism. We describe here the first structure of a ternary R67 DHFR.DHF.NADP+ catalytic complex, resolved to 1.26 A. This structure provides the first clear picture of how this enzyme, which lacks the active site carboxyl residue that is ubiquitous in Type I DHFRs, is able to function. In the catalytic complex, the polar backbone atoms of two symmetry-related I68 residues provide recognition motifs that interact with the carboxamide on the nicotinamide ring, and the N3-O4 amide function on the pteridine ring. This set of interactions orients the aromatic rings of substrate and cofactor in a relative endo geometry in which the reactive centers are held in close proximity. Additionally, a central, hydrogen-bonded network consisting of two pairs of Y69-Q67-Q67'-Y69' residues provides an unusually tight interface, which appears to serve as a "molecular clamp" holding the substrates in place in an orientation conducive to hydride transfer. In addition to providing the first clear insight regarding how this extremely unusual enzyme is able to function, the structure of the ternary complex provides general insights into how a mutationally challenged enzyme, i.e., an enzyme whose evolution is restricted to four-residues-at-a-time active site mutations, overcomes this fundamental limitation.

Role of the catalytic metal during polymerization by DNA polymerase lambda.

The incorporation of dNMPs into DNA by polymerases involves a phosphoryl transfer reaction hypothesized to require two divalent metal ions. Here we investigate this hypothesis using as a model human DNA polymerase lambda (Pol lambda), an enzyme suggested to be activated in vivo by manganese. We report the crystal structures of four complexes of human Pol lambda. In a 1.9 A structure of Pol lambda containing a 3'-OH and the non-hydrolyzable analog dUpnpp, a non-catalytic Na+ ion occupies the site for metal A and the ribose of the primer-terminal nucleotide is found in a conformation that positions the acceptor 3'-OH out of line with the alpha-phosphate and the bridging oxygen of the pyrophosphate leaving group. Soaking this crystal in MnCl2 yielded a 2.0 A structure with Mn2+ occupying the site for metal A. In the presence of Mn2+, the conformation of the ribose is C3'-endo and the 3'-oxygen is in line with the leaving oxygen, at a distance from the phosphorus atom of the alpha-phosphate (3.69 A) consistent with and supporting a catalytic mechanism involving two divalent metal ions. Finally, soaking with MnCl2 converted a pre-catalytic Pol lambda/Na+ complex with unreacted dCTP in the active site into a product complex via catalysis in the crystal. These data provide pre- and post-transition state information and outline in a single crystal the pathway for the phosphoryl transfer reaction carried out by DNA polymerases.

Identification and functional characterization of polymorphisms in human cyclooxygenase-1 (PTGS1).

OBJECTIVE: Cyclooxygenase-1 (COX-1, PTGS1) catalyzes the conversion of arachidonic acid to prostaglandin H2, which is subsequently metabolized to various biologically active prostaglandins. We sought to identify and characterize the functional relevance of genetic polymorphisms in PTGS1. METHODS: Sequence variations in human PTGS1 were identified by resequencing 92 healthy individuals (24 African, 24 Asian, 24 European/Caucasian, and 20 anonymous). Using site-directed mutagenesis and a baculovirus/insect cell expression system, recombinant wild-type COX-1 and the R8W, P17L, R53H, R78W, K185T, G230S, L237M, and V481I variant proteins were expressed. COX-1 metabolic activity was evaluated in vitro using an oxygen consumption assay under basal conditions and in the presence of indomethacin. RESULTS: Forty-five variants were identified, including seven nonsynonymous polymorphisms encoding amino acid substitutions in the COX-1 protein. The R53H (35+/-5%), R78W (36+/-4%), K185T (59+/-6%), G230S (57+/-4%), and L237M (51+/-3%) variant proteins had significantly lower metabolic activity relative to wild-type (100+/-7%), while no significant differences were observed with the R8W (104+/-10%), P17L (113+/-7%), and V481I (121+/-10%) variants. Inhibition studies with indomethacin demonstrated that the P17L and G230S variants had significantly lower IC50 values compared to wild-type, suggesting these variants significantly increase COX-1 sensitivity to indomethacin inhibition. Consistent with the metabolic activity data, protein modeling suggested the G230S variant may disrupt the active conformation of COX-1. CONCLUSIONS: Our findings demonstrate that several genetic variants in human COX-1 significantly alter basal COX-1-mediated arachidonic acid metabolism and indomethacin-mediated inhibition of COX-1 activity in vitro. Future studies characterizing the functional impact of these variants in vivo are warranted.

Magnesium-induced assembly of a complete DNA polymerase catalytic complex.

The molecular details of the nucleotidyl transferase reaction have remained speculative, as strategies to trap catalytic intermediates for structure determination utilize substrates lacking the primer terminus 3'-OH and catalytic Mg2+, resulting in an incomplete and distorted active site geometry. Since the geometric arrangement of these essential atoms will impact chemistry, structural insight into fidelity strategies has been hampered. Here, we present a crystal structure of a precatalytic complex of a DNA polymerase with bound substrates that include the primer 3'-OH and catalytic Mg2+. This catalytic intermediate was trapped with a nonhydrolyzable deoxynucleotide analog. Comparison with two new structures of DNA polymerase beta lacking the 3'-OH or catalytic Mg2+ is described. These structures provide direct evidence that both atoms are required to achieve a proper geometry necessary for an in-line nucleophilic attack of O3' on the alphaP of the incoming nucleotide.

Structural analysis of strand misalignment during DNA synthesis by a human DNA polymerase.

Insertions and deletions in coding sequences can alter the reading frame of genes and have profound biological consequences. In 1966, Streisinger proposed that these mutations result from strand slippage, which in repetitive sequences generates misaligned intermediates stabilized by correct base pairing that support polymerization. We report here crystal structures of human DNA polymerase lambda, which frequently generates deletion mutations, bound to such intermediates. Each contains an extrahelical template nucleotide upstream of the active site. Surprisingly, the extra nucleotide, even when combined with an adjacent mismatch, does not perturb polymerase active site geometry, which is indistinguishable from that for correctly aligned strands. These structures reveal how pol lambda can polymerize on substrates with minimal homology during repair of double-strand breaks and represent strand-slippage intermediates consistent with Streisinger's classical hypothesis. They are thus relevant to the origin of single-base deletions, a class of mutations that can confer strong biological phenotypes.

Structural insight into the DNA polymerase beta deoxyribose phosphate lyase mechanism.

A large number of biochemical and genetic studies have demonstrated the involvement of DNA polymerase beta (Pol beta) in mammalian base excision repair (BER). Pol beta participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5'-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol beta complexed with 5'-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1' of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120 degrees about the dRP 3'-phosphate is required to position the epsilon-amino Lys72 close to the dRP C1'. This model also suggests that several other side chains are in position to facilitate the beta-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity.

Crystal structures, reactivity and inferred acylation transition states for 2'-amine substituted RNA.

Ribose 2'-amine substitutions are broadly useful as structural probes in nucleic acids. In addition, structure-selective chemical reaction at 2'-amine groups is a robust technology for interrogating local nucleotide flexibility and conformational changes in RNA and DNA. We analyzed crystal structures for several RNA duplexes containing 2'-amino cytidine (C(N)) residues that form either C(N)-G base pairs or C(N)-A mismatches. The 2'-amine substitution is readily accommodated in an A-form RNA helix and thus differs from the C2'-endo conformation observed for free nucleosides. The 2'-amide product structure was visualized directly by acylating a C(N)-A mismatch in intact crystals and is also compatible with A-form geometry. To visualize conformations able to facilitate formation of the amide-forming transition state, in which the amine nucleophile carries a positive partial charge, we analyzed crystals of the C(N)-A duplex at pH 5, where the 2'-amine is protonated. The protonated amine moves to form a strong electrostatic interaction with the 3'-phosphodiester. Taken together with solution-phase experiments, 2'-amine acylation is likely facilitated by either of two transition states, both involving precise positioning of the adjacent 3'-phosphodiester group.