Secreted Proteases Control Autolysin-mediated Biofilm Growth of Staphylococcus aureus

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.     

Anatomical characterization of Strobilanthes (Acanthaceae) species from the northern Western Ghats of India and its implication for identification at vegetative state

A comprehensive study of stem, leaf and petiole anatomy of 10 species of Strobilanthes from northern Western Ghats of India was carried out to identify characteristics which would enable species identification when flowering material is unavailable. In Strobilanthes, some species bloom annually, others are plietesials, i.e. they grow without blooming for several years and then produce huge quantities of flowers, release seeds and die. Therefore, alternative methods, such as anatomical characters, are essential to distinguish Strobilanthes species in their vegetative stage. We collected ten species of Strobilanthes for anatomical characterization. Under the bright‐field microscope, stem cross‐sections of different species were found to be undulate, quadrangular, quadrangular‐winged or terete. Study of the stem revealed a distinct outer and inner cortex, the distribution of cystoliths (CaCO₃ crystals), raphides (CaC₂H₂O₅ crystals) and sclereids which varied from species to species. Study of leaf anatomy showed structural variation and vascular bundle shapes that differed between the species. Leaf epidermal characters under light and scanning electron microscopy exhibited variation in characters such as stomatal index, stomatal length and width, stomatal type and presence of glandular and non‐glandular trichomes. The petiole anatomy was species‐specific, especially with respect to vascular bundle structure and the distribution of structures such as sclereids, cystoliths, sphaeraphides and tannin cells varied. Hence, unique anatomical features of the stem, leaf and petiole could be used as taxonomic characters to identify Strobilanthes species in a vegetative state.     

The crystal structure of C2a, the catalytic fragment of classical pathway C3 and C5 convertase of human complement

The multi-domain serine protease C2 provides the catalytic activity for the C3 and C5- convertases of the classical and lectin pathways of complement activation. Formation of these convertases requires the Mg(2+)-dependent binding of C2 to C4b, and the subsequent cleavage of C2 by C1s or MASP2, respectively. The C-terminal fragment C2a consisting of a serine protease (SP) and a von Willebrand factor type A (vWFA) domain, remains attached to C4b, forming the C3 convertase, C4b2a. Here, we present the crystal structure of Mg(2+)-bound C2a to 1.9 A resolution in comparison to its homolog Bb, the catalytic subunit of the alternative pathway C3 convertase, C3bBb. Although the overall domain arrangement of C2a is similar to Bb, there are certain structural differences. Unexpectedly, the conformation of the metal ion-dependent adhesion site and the position of the alpha7 helix of the vWFA domain indicate a co-factor-bound or open conformation. The active site of the SP domain is in a zymogen-like inactive conformation. On the basis of these structural features, we suggest a model for the initial steps of C3 convertase assembly.     

Geometries and electronic structures of cyanide adducts of the non-heme iron active site of superoxide reductases: vibrational and ENDOR studies

We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide and have used vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Addition of cyanide changes the active site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13CN, C15N, and 13C15N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle approximately 175 degrees) and two distinct bent conformations (Fe-C-N angles <140 degrees). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state, indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13C-ENDOR spectra for the 13CN-bound ferric active sites in both 1Fe- and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123 degrees (C3) and 133 degrees (C2), taking a reference value from vibrational studies of 175 degrees (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.     

Mitochondrial replication origin stability and propensity of adjacent tRNA genes to form putative replication origins increase developmental stability in lizards

Secondary structure stability of mitochondrial origins of light-strand replication (OL) presumably reduces delayed formation of light-strand initiating replication forks on the heavy strand. Delayed replication initiation prolongs single strandedness of the heavy strand. More mutations accumulate during the prolonged time spent single stranded. Presumably, delayed replication initiation and excess mutations affect mitochondrial biochemical processes and ultimately morphological outcomes of development at the whole-organism level. This predicts that developmental stability increases with OL secondary structure stability and with formation of OL-like structures by the five tRNA genes flanking recognized OLs. Stable OLs and high percentages of OL-resembling secondary structures of adjacent tRNA genes (predicted by Mfold) correlate positively with developmental stability in three lizard families (Anguidae, Amphisbaenidae, and Polychrotidae). Accounting for effects of the regular OL, Sfold-predicted OL-like propensity of the entire tRNA gene cluster (not of individual genes) correlates with increased developmental stability in Anguidae, also across the entire free-energy range of Boltzmann's distribution of secondary structures. In the fossorial Amphisbaenidae, the OL-like structure-forming propensity of tRNA genes correlates positively with developmental stability for the distribution's sub-optimally stable regions, and negatively for its optimally stable regions, suggesting the thermoregulated functioning of OL vs. flanking tRNA genes as replication origins. Results for polychrotid tRNA genes are intermediate. Anguid tRNA genes possibly function in addition to the regular OL. Mitochondrial tRNA genes may thus frequently acquire and lose the alternative OL function, without sequence (gene) duplication and loss of their primary function.     

Cat lung hemodynamics: comparison of experimental results and model predictions

Commonly, attempts have been made to learn about the structure and function of the pulmonary vascular bed from measurements of arterial and venous pressures and blood flow rate under steady-state conditions (e.g., from pressure vs. flow data) or dynamic conditions (e.g., from vascular occlusion data). Zhuang et al. (J. Appl. Physiol. 55: 1341-1348, 1983) have presented a detailed model of steady-state cat lung hemodynamics based on direct measurements of anatomical and elasticity data. This model provides an opportunity to better understand the information content of the hemodynamic data. Therefore, in the present study we carried out a series of steady-state and dynamic experiments on isolated cat lungs. We then compared the results with those predicted by the model. We found that the model provided a good fit to the steady-state data. However, to fit the dynamic data, some modifications were necessary to account for the viscous behavior of the vessel walls and to move the first moment of the distribution of vascular resistance toward the arterial end of the vascular bed relative to that of the distribution of vascular compliance. Due to the sensitivity of the vascular resistance to small changes in vessel diameters and branching ratio, the modifications in morphometry represent small changes in morphometric data and are probably within the range of uncertainty in such data. The modifications had little effect on the steady-state model simulations but substantially improved the dynamic model simulations, suggesting that the dynamic data are quite sensitive to small changes in the relative distributions of vessel diameters and elasticity.     

Rice caryopsis structure in relation to distribution of micronutrients (iron, zinc, β-carotene) of rice cultivars including transgenic indica rice

The transgenic indica rice lines of IR68144 and BR29, developed using endosperm-specific promoters were analyzed for their iron, zinc and β-carotene content in the endosperm. Biochemical analysis clearly revealed the presence of higher accumulation of iron, zinc and β-carotene in transgenic rice grains in comparison with control. Prussian blue staining reaction evidenced the presence of iron in the endosperm cells of transgenic rice grains in comparison with control where iron is restricted only to aleurone and embryo. The rice grain structure of IR64, IR72, IR68144, Swarna, BRRI Dhan 29 (BR29), BR28, Taipai 309 (T309) and New Plant Type-3 (NPT3) indicated that the number of aleurone layers, size of the embryo and size of the caryopsis determines the quantity of important micronutrients (iron, zinc) in the grains. Biochemical analysis revealed that iron and zinc content drastically varies in polished and unpolished rice and among the varieties examined. During the polishing process almost entire aleurone and most part of the embryo is removed which are the main storehouse for major micronutrients. It is estimated that more than 70% of micronutrients are lost during polishing process.     

Uncovering directional sensing: where are we headed?

We survey aspects of directional sensing, i.e. how a cell interprets differences in the external concentration of a chemoattractant to guide its motion, from the perspective of systems biology. We focus on questions that need to be addressed using a combination of modelling and experimental approaches. After briefly summarising the ideas underlying recent modelling efforts, we discuss a variety of experimental questions which are motivated by these models. Some of these questions focus on basic features of the chemotactic response, without involving much biochemistry, while others focus on filling some of the gaps in the biochemistry, which have been brought to light by the models. The emphasis is on systematic quantitative experiments that will unambiguously resolve many of these issues. Finally, we describe some current challenges for theoretical modelling and survey some of the theoretical tools and approaches employed to model the chemotaxis pathways.     

Disease-associated polyglutamine stretches in monomeric huntingtin adopt a compact structure

Abnormal polyglutamine (polyQ) tracts are the only common feature in nine proteins that each cause a dominant neurodegenerative disorder. In Huntington's disease, tracts longer than 36 glutamines in the protein huntingtin (htt) cause degeneration. In situ, monoclonal antibody 3B5H10 binds to different htt fragments in neurons in proportion to their toxicity. Here, we determined the structure of 3B5H10 Fab to 1.9?? resolution by X-ray crystallography. Modeling demonstrates that the paratope forms a groove suitable for binding two β-rich polyQ strands. Using small-angle X-ray scattering, we confirmed that the polyQ epitope recognized by 3B5H10 is a compact two-stranded hairpin within monomeric htt and is abundant in htt fragments unbound to antibody. Thus, disease-associated polyQ stretches preferentially adopt compact conformations. Since 3B5H10 binding predicts degeneration, this compact polyQ structure may be neurotoxic.