Structural evidence for the evolution of pyrogenic toxin superantigens.

Pathogenic bacteria have evolved a wide variety of toxins to invade and attack host organisms. In particular, strains of the bacteria Staphylococcus aureus and Streptococcus pyogenes produce a family of pyrogenic toxin superantigens (PTSAgs) that can cause illness, e.g., toxic shock syndrome, or synergize with a number of other immune system disorders. The PTSAgs are all similar in size and have a conserved two-domain tertiary fold despite minimal amino acid sequence identity. The tertiary structure of PTSAg domain 1 is similar to the immunoglobulin binding motif of streptococcal proteins G and L. PTSAg domain 2 resembles members of the oligosaccharide/oligonucleotide binding fold family that includes the B subunits of the AB(5) heat-labile enterotoxins, cholera toxin, pertussis toxin, and verotoxin. The strong structural homology between the pyrogenic toxins and other bacterial proteins suggests that the PTSAgs evolved through the recombination of two smaller beta-strand motifs.

Disruption of the pelvic ring in pediatric patients.

Pelvic ring disruptions are rare in children. Generally, nonoperative treatment has been recommended for children with these injuries. The authors retrospectively reviewed the medical records and radiographs of 189 patients with pelvic ring disruptions who were evaluated at a tertiary care children's hospital during a 10-year period. Of the 189 patients, 57 were identified with unstable pelvic fractures. These fractures were classified according to Tile, and Torode and Zieg. Forty-three of these patients were available to complete a subjective evaluation of their treatment at midterm followup. Thirteen patients were treated operatively and 30 were treated nonoperatively. There was no significant difference in subjective scoring between the two groups for pain at rest, pain with activity, limp, leg length discrepancy, and overall satisfaction with treatment. Overall, there were 92% good or excellent results in the patients who were treated operatively and 80% good or excellent results in the patients who were treated nonoperatively. Patients in both groups reported a high level of satisfaction with their outcome regardless of their fracture classification and treatment method.

Pyrogenic toxin superantigen site specificity in toxic shock syndrome and food poisoning in animals.

Staphylococcus aureus and Streptococcus pyogenes express pyrogenic toxin superantigens (PTSAgs) that are associated with toxic shock syndrome (TSS) and staphylococcal food poisoning (SFP). Most PTSAgs cause TSS in deep-tissue infections, whereas only TSS toxin 1 (TSST-1) is associated with menstrual, vaginal TSS. In contrast, SFP has been linked only with staphylococcal enterotoxins (SEs). Because of the differential abilities of PTSAgs to cause systemic or localized symptoms in a site-dependent manner, the present study was undertaken to assess the toxins' abilities to cross mucosal barriers. The activity of three PTSAgs when delivered orally, vaginally, or intravenously to rabbits and orally to monkeys was investigated. TSST-1 induced shock via all three routes in rabbits. Although active when administered intravenously, SEC1 and streptococcal pyrogenic exotoxin A (SPEA) did not cause symptoms when administered orally or vaginally. Only SEC1 induced emesis in the monkey feeding assay. TSST-1, albeit less stable than SEC1 and SPEA to pepsin, induced diarrhea in monkeys. Our results may explain the unique association of TSST-1 with menstrual TSS and why SPEA is only rarely associated with TSS after pharyngitis, despite being highly associated with TSS after subcutaneous infections. Finally, our studies indicate that enterotoxicity in SFP is not the result of superantigenicity.

Crystal structure of the truncated cubic core component of the Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex.

The dihydrolipoamide succinyltransferase (E2o) component of the 2-oxoglutarate dehydrogenase multienzyme complex is composed of 24 subunits arranged with 432 point group symmetry. The catalytic domain (CD) of the E2o component catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. The crystal structure of the Escherichia coli E2oCD has been solved to 3.0 A resolution using molecular replacement phases derived from the structure of the catalytic domain from the Azotobacter vinelandii dihydrolipoamide acetyltransferase (E2pCD). The refined model of the E. coli E2oCD consists of residues 172 to 404 and has an R-factor of 0.205 (Rfree=0.249) for 9696 reflections between 20.0 and 3.0 A resolution. Although both E2oCD and E2pCD form 24mers, subtle changes in the orientations of two helices in E2oCD increase the stability of the E2oCD 24mer in comparison to the less stable A. vinelandii E2pCD 24mer. Like E2pCD and chloramphenicol acetyltransferase (CAT), the active site of E2oCD is located in the middle of a channel formed at the interface between two 3-fold related subunits. Two of the active-site residues (His375 and Thr323) have a similar orientation to their counterparts in E2pCD and CAT. A third catalytic residue (Asp379) assumes a conformation similar to the corresponding residue in E2pCD (Asn614), but different from its counterpart in CAT (Asp199). Binding of the substrates to E2oCD is proposed to induce a change in the conformation of Asp379, allowing this residue to form a salt bridge with Arg184 that is analogous to that formed between Asp199 and Arg18 in CAT. Computer models of the active site of E2o complexed with dihydrolipoamide and with coenzyme A led to the identification of the probable succinyl-binding pocket. The residues which form this pocket (Ser330, Ser333, and His348) are probably responsible for E2o's substrate specificity.

Structures of five mutants of toxic shock syndrome toxin-1 with reduced biological activity.

The three-dimensional structures of five mutants of toxic shock syndrome toxin-1 (TSST-1) have been determined. These mutations are in the long central alpha helix and are useful in mapping portions of TSST-1 involved in superantigenicity and lethality. The T128A, H135A, Q139K, and I140T mutations appear to reduce superantigenicity by altering the properties of the T-cell receptor interaction surface. The Q136A mutation is at a largely buried site and causes a dramatic change in the conformation of the beta7-beta9 loop which covers the back of the central alpha helix. As this mutation has the unique ability to reduce the toxin's lethality in rabbits while retaining its superantigenicity, it raises the possibility that this rear loop mediates the ability of TSST-1 to induce lethality and suggests a route for producing nonlethal toxins for therapeutic development.

Peer reviewed: nanomaterials in analytical chemistry.

As particle size approaches molecular dimensions, all properties of a material change, making nanomaterials useful for particular applications.

Refined structures of three crystal forms of toxic shock syndrome toxin-1 and of a tetramutant with reduced activity.

The structure of toxic shock syndrome toxin-1 (TSST-1), the causative agent in toxic shock syndrome, has been determined in three crystal forms. The three structural models have been refined to R-factors of 0.154, 0.150, and 0.198 at resolutions of 2.05 A, 2.90 A, and 2.75 A, respectively. One crystal form of TSST-1 contains a zinc ion bound between two symmetry-related molecules. Although not required for biological activity, zinc dramatically potentiates the mitogenicity of TSST-1 at very low concentrations. In addition, the structure of the tetramutant TSST-1H [T69I, Y80W, E132K, I140T], which is nonmitogenic and does not amplify endotoxin shock, has been determined and refined in a fourth crystal form (R-factor = 0.173 to 1.9 A resolution).

Development and validation of the Cholesterol-Saturated Fat Index (CSI) Scorecard: a dietary self-monitoring tool.

OBJECTIVE: The Cholesterol-Saturated Fat Index (CSI) Scorecard was developed and tested as a self-monitoring tool for patients consuming a cholesterol-lowering diet. SUBJECTS/DESIGN: Twelve nutritionists used the CSI Scorecard to estimate the CSI score of the same five food records. Subsequently, using a revised tool, 12 subjects with hypercholesterolemia estimated the CSI score of their 4-day food records and 11 participated in evaluation meetings. STATISTICAL ANALYSES: Two-tailed, one-sample t tests and the Spearman rho correlation test were used, respectively, to compare CSI Scorecard estimates of nutritionists and participants to CSI scores obtained from computer calculations. RESULTS: The CSI scores estimated by the nutritionists were close to the computed scores at the 25th and 75th food record percentiles. The correlation of the estimated CSI scores of participants to the computed scores was rs = .8 (P < .05). APPLICATION: The CSI Scorecard is a new, simple, quick, and accurate dietary self-monitoring tool patients can use in research centers and primary care settings. By numerically illustrating the saturated fat and cholesterol content of foods, the CSI Scorecard facilitates dietary self-monitoring and, thus, self-efficacy in the adoption and maintenance of a cholesterol-lowering dietary pattern.

Localization of biologically important regions on toxic shock syndrome toxin 1.

Toxic shock syndrome toxin 1 (TSST-1) contains a long central alpha helix that forms the base of two grooves on opposite sides of the molecule. Previous studies indicated that residues 132, 135, and 140 along the back of the central alpha helix are important in the biological activities. We made mutations of additional central alpha-helix residues exposed along this groove on the back of TSST-1. The proteins were purified, shown not to have gross alteration in structure, and tested for both superantigenicity and ability to elicit lethal TSS, using the superantigenicity, likely to because of alteration in T-cell receptor binding. Mutants H135A, Q136A, and E132K/ Q136K lost the ability to induce lethal TSS. The mutant Q136A was most increasing because it was superantigenic, yet nonlethal.

Photosynthesis and nutrient-use efficiency of barley in response to low arbuscular mycorrhizal colonization and addition of phosphorus.

The effects of arbuscular mycorrhizal infection by Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe on growth and photosynthesis of barley (Hordeum vulgare L. cv. Manitou) were investigated in sand culture at five levels of calcium phosphate (50, 100, 200, 400 and 800 mg P kg(-1) ). Mycorrhizal infection was low and varied with P supply, declining from 3.3% at 50 mg P kg(-1) to 1.5% at the highest P concentration. In general, there were small differences in biomass between mycorrhizal (+AM) and non-mycorrhizal (-AM) barley but a significant reduction in dry mass of senesced leaves occurred in the +AM plants. Leaf P concentrations increased with P application, but did not differ between + AM and -AM plants. Although there were no differences in dry mass between + AM and -AM plants at 50 mg P kg(-1) , it was at this lowest P supply that +AM plants had higher rates of photosynthesis and greater P-use and N-use efficiencies. The mycorrhizal enhancement of maximum photosynthetic rate at the lowest P level was associated with a higher stomatal conductance, but was not related to increased leaf P or to changes in photon yield or the ratio of variable (FV) to maximum (FM) chlorophyll fluorescence.

Three-dimensional structure of a hemichrome hemoglobin from Caudina arenicola.

The structure of a monomeric hemichrome form of an invertebrate hemoglobin, Hb-C chain, from Caudina arenicola has been refined to an R value of 0.16 using the data from 5.0 to 2.5 A resolution (R = 0.21 from 10.0 to 2.5 A resolution). Hb-C crystallizes in space group P2(l) with cell constants a = 45.74, b = 45.23 and c = 40.92 A and beta = 104.4 degrees with two monomers packed in the unit cell (V(m) = 2.34 A(3) Da(-1)). The phases were determined by the multiple isomorphous replacement method with Hg(2+) the major derivative. The structure consists of 157 amino acids with N- and C-terminal regions and eight alpha-helices forming a heme pocket. The unique feature of this structure is the hemichrome form with the proximal and distal histidines coordinated to the heme Fe atom, which is nearly in the plane of the porphyrin ring. A total of 111 solvent molecules were added to the structure using difference density peaks of at least 3sigma over background. Interestingly, all the heme groups present in the crystal are nearly coplanar.

X-ray structure determination of a dimeric hemoglobin from Caudina arenicola.

The X-ray structure of a dimeric, cyanomet-liganded hemoglobin D-chain (Hb-D) from Caudina arenicola has been determined by the molecular-replacement method. The search model was a concatenated model of three hemoglobin structures superimposed on the backbone of monomeric, hemichrome hemoglobin C-chain (Hb-C) from the same organism. Hb-D crystallizes in space group P4(1)2(1)2 with cell constants a = b = 77.0 and c =61.5 A with one subunit in the asymmetric unit. The dimer twofold axis corresponds to a crystallographic twofold along one of the body diagonals of the unit cell. Rotation and translation searches as well as model refinement were carried out in X-PLOR with the final model having an R value of 0.19 using the data from 5.0 to 2.9 A resolution (R = 0.26 for 10.0 to 2.9 A resolution). The homodimeric structure of Caudina Hb-D features close heme-heme contacts with an Fe-Fe distance of 19.0 A. The subunit-subunit interface involves both the E and F helices from each subunit with the E helices oriented antiparallel at 50 degrees with respect to one another, similar to the quaternary structure observed for the homodimeric hemoglobin from Scapharca inaequivalvis.

Structural analysis of monomeric hemichrome and dimeric cyanomet hemoglobins from Caudina arenicola.

The X-ray structures of two hemoglobins (Hb) from the sea cucumber Caudina arenicola (an echinoderm) have been determined: a low spin, hemichrome, monomeric Hb-C chain, and a cyanomet-liganded dimeric Hb-D chain. Attempts to obtain crystal structures of the deoxy-liganded and hemichrome forms from the same chain type have not been successful. In this work, the Hb-C chain and Hb-D chain structures are compared, and differences observed in tertiary structure related to the different ligand states for hemoglobin chains from this organism. In addition to shifts of the distal histidine and E helix, differences are noted in the position of the heme group within the heme pocket, the hydrogen bonding of the heme group to the protein, and the status of the D helix. These differences are important in understanding the ligand-linked association states of these hemoglobins. The quaternary structure of the Hb-D homodimer is compared with those from two other invertebrate hemoglobins from Scapharca inaequivalvis and Urechis caupo, which also have subunit-subunit interactions that involve the E and E' helices. The dimer interactions of the Caudina and Urechis hemoglobins are quite dissimilar. However, the dimer interface observed in cyanomet Hb-D is strikingly similar to that observed for the carbonmonoxy hemoglobin dimer from the clam, Scapharca, yet many of the key amino acid residues implicated in the cooperative mechanism of the Scapharca hemoglobin are not conserved in the Caudina hemoglobins.

Aspects of the reproductive ecology of four australian Hakea species (Proteaceae) in South Africa.

Four shrub species of the Australian Proteaceae (Hakea sericea, H. gibbosa, H. suaveolens and H. salicifolia) were introduced to South African fynbos shrublands between 1840 and 1860. H. sericea is highly invasive, H. gibbosa and H. suaveolens are moderately invasive and H. salicifolia is not invasive. The allocation of reproductive energy, germinability, the ability to survive fires and to germinate in burnt and unburnt areas, and the nutrient content of seeds were assessed for the four species. The information was used to investigate whether the success of H. sericea relative to the other three species could be explained by the superior expression of any trait. The most important trait which separates H. sericea from the other species is its ability to produce a large seed bank in its adopted environment in the absence of seed predators. Seed production in H. sericea shrubs with an above-ground dry mass of 8 kg is four times greater than H. gibbosa and more than 16 times that of H. suaveolens. Although H. salicifolia also produces a large seed bank, its seeds are unable to survive fires due to inadequate insulation by the small follicles. The results are compared to dispersal and seed bank data for indigenous South African Proteaceae, which have low dispersal and suffer high pre-dispersal seed predation. We suggest that potential invasives in the fynbos can be identified as species that have: (i) a potentially high seed production that is limited by specialized predators; (ii) an ability to disperse over long distances; and (iii) are pre-adapted to frequent fires and low soil nutrients. The data also support the current strategy of combatting H. sericea using specialized insect seed predators.