Petrobactin is the Primary Siderophore Synthesized by <Emphasis Type="BoldItalic">Bacillus anthracis</Emphasis> Str. <Emphasis Type="BoldItalic">Sterne</Emphasis> under Conditions of Iron Starvation

The siderophores of Bacillus anthracis are critical for the pathogen’s proliferation and may be necessary for its virulence. Bacillus anthracis str. Sterne cells were cultured in iron free media and the siderophores produced were isolated and purified using a combination of XAD-2 resin, reverse-phase FPLC, and size exclusion chromatography. A combination of ¹H and ¹³C NMR spectroscopy, UV spectroscopy and ESI-MS/MS fragmentation were used to identify the primary siderophore as petrobactin, a catecholate species containing unusual 3,4-dihydroxybenzoate moieties, previously only identified in extracts of Marinobacter hydrocarbonoclasticus. A secondary siderophore was observed and structural analysis of this species is consistent with that reported for bacillibactin, a siderophore observed in many species of bacilli. This is the first structural characterization of a siderophore from B. anthracis, as well as the first characterization of a 3,4-DHB containing catecholate in a pathogen.     

The Influence of Diet Composition on Fitness of the Blue Crab,Callinectes sapidus

The physiological condition and fecundity of an organism is frequently controlled by diet. As changes in environmental conditions often cause organisms to alter their foraging behavior, a comprehensive understanding of how diet influences the fitness of an individual is central to predicting the effect of environmental change on population dynamics. We experimentally manipulated the diet of the economically and ecologically important blue crab, Callinectes sapidus, to approximate the effects of a dietary shift from primarily animal to plant tissue, a phenomenon commonly documented in crabs. Crabs whose diet consisted exclusively of animal tissue had markedly lower mortality and consumed substantially more food than crabs whose diet consisted exclusively of seaweed. The quantity of food consumed had a significant positive influence on reproductive effort and long-term energy stores. Additionally, seaweed diets produced a three-fold decrease in hepatopancreas lipid content and a simultaneous two-fold increase in crab aggression when compared to an animal diet. Our results reveal that the consumption of animal tissue substantially enhanced C. sapidus fitness, and suggest that a dietary shift to plant tissue may reduce crab population growth by decreasing fecundity as well as increasing mortality. This study has implications for C. sapidus fisheries.     

The exoskeletal structure and tensile loading behavior of an ant neck joint

Insects have evolved mechanical form and function over millions of years. Ants, in particular, can lift and carry heavy loads relative to their body mass. Loads are lifted with the mouthparts, transferred through the neck joint to the thorax, and distributed over six legs and tarsi (feet) that anchor to the supporting surface. While previous research has explored attachment mechanisms of the tarsi, little is known about the relation between the mechanical function and the structural design and material properties of the ant. This study focuses on the neck – the single joint that withstands the full load capacity. We combine mechanical testing, computed tomography (CT), scanning electron microscopy (SEM), and computational modeling to better understand the mechanical structure–function relation of the neck joint of the ant species Formica exsectoides (Allegheny mound ant). Our mechanical testing results show that the soft tissue forming the neck joint of F. exsectoides exhibits an elastic modulus of 230±140 MPa and can withstand ~5000 times the ant's weight. We developed a 3-dimensional (3D) model of the structural components of the neck joint for simulation of mechanical behavior. Finite element (FE) simulations reveal the neck-to-head transition where the soft membrane material meets the hard exoskeleton as the critical point for failure of the neck joint, which is consistent with our experiments. Our results further indicate that the neck joint structure exhibits anisotropic mechanical behavior with the highest stiffness occurring when the load path is aligned with the axis of the neck.     

Isolation and sequence determination of peptide components of atrial natriuretic factor

The independent isolation and sequence determination in our laboratories of three closely related Atrial Natriuretic Factor peptides from rat atria confirm the sequences of ANF peptides reported by Seidah et al and synthesized by Nutt et al [Proc. Natl. Acad. Sci., (1984) in press] and contain the sequences reported by Flynn et al [Biochem. Biophys. Res. Commun. (1983) 117: 859-865] and by Currie et al [Science (1984) 223: 67-69]. In addition, we provide proof for a C-terminal tyrosine rather than tyrosine amide in our isolated peptides.     

Novel Insights into Eukaryotic γ-Glutamyltranspeptidase 1 from the Crystal Structure of the Glutamate-bound Human Enzyme

The enzyme γ-glutamyltranspeptidase 1 (GGT1) is a conserved member of the N-terminal nucleophile hydrolase family that cleaves the γ-glutamyl bond of glutathione and other γ-glutamyl compounds. In animals, GGT1 is expressed on the surface of the cell and has critical roles in maintaining cysteine levels in the body and regulating intracellular redox status. Expression of GGT1 has been implicated as a potentiator of asthma, cardiovascular disease, and cancer. The rational design of effective inhibitors of human GGT1 (hGGT1) has been delayed by the lack of a reliable structural model. The available crystal structures of several bacterial GGTs have been of limited use due to differences in the catalytic behavior of bacterial and mammalian GGTs. We report the high resolution (1.67 Å) crystal structure of glutamate-bound hGGT1, the first of any eukaryotic GGT. Comparisons of the active site architecture of hGGT1 with those of its bacterial orthologs highlight key differences in the residues responsible for substrate binding, including a bimodal switch in the orientation of the catalytic nucleophile (Thr-381) that is unique to the human enzyme. Compared with several bacterial counterparts, the lid loop in the crystal structure of hGGT1 adopts an open conformation that allows greater access to the active site. The hGGT1 structure also revealed tightly bound chlorides near the catalytic residue that may contribute to catalytic activity. These are absent in the bacterial GGTs. These differences between bacterial and mammalian GGTs and the new structural data will accelerate the development of new therapies for GGT1-dependent diseases.     

Predator size interacts with habitat structure to determine the allometric scaling of the functional response

While both predator body size and prey refuge provided by habitat structure have been established as major factors influencing the functional response (per capita consumption rate as a function of prey density), potential interactions between these factors have rarely been explored. Using a crab predator (Panopeus herbstii) – mussel prey (Brachidontes exustus) system, we examined the allometric scaling of the functional response in oyster (Crassostrea virginica) reef habitat, where crevices within oyster clusters provide mussels refuge from predation. A field survey of mussel distribution showed that mussels attach closer to the cluster periphery at high mussel density, indicating the potential for saturation of the refuge. In functional response experiments, the consumption rate of large crabs was depressed at low prey density relative to small crabs, while at high prey density the reverse was true. Specifically, the attack rate coefficient and handling time both decreased non‐linearly with crab size. An additional manipulation revealed that at low prey densities, the ability of large crabs to maneuver their claws and bodies to extract mussels from crevices was inhibited relative to small crabs by the structured habitat, reducing their attack rate. At high prey densities, crevices were saturated, forcing mussels to the edge of clusters where crabs were only limited by handling time. Our study illuminates a potentially general mechanism where the quality of the prey refuge provided by habitat structure is dependent on the relative size of the predator. Thus anthropogenic influences that alter the natural crab size distribution or degrade reef habitat structure could threaten the long‐term stability of the crab –mussel interaction in reefs.     

Cytokines regulate cysteine cathepsins during TLR responses

TLR activation is an important component of innate immunity but also contributes to the severity of inflammatory diseases. Cysteine cathepsins (Cat) B, L and S, which are endosomal and lysosomal proteases, participate in numerous physiological systems and are upregulated during various inflammatory disorders and cancers. Macrophages have the highest cathepsin expression and are major contributors to inflammation and tissue damage during chronic inflammatory diseases. We investigated the impact of TLR activation on macrophage Cat B, L and S activities using live-cell enzymatic assays. TLR2, TLR3 and TLR4 ligands increased intracellular activities of these cathepsins in a differential manner. TLR4-induced cytokines increased proteolytic activities without changing mRNA expression of cathepsins or their endogenous inhibitors. Neutralizing antibodies recognizing TNF-α, IL-1β and IFN-β differentially eliminated cathepsin upregulation. These findings indicate cytokines induced by MyD88-dependent and -independent signaling cascades regulate cathepsin activities during macrophage responses to TLR stimulation.