Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

In this report we investigated, within a group of closely related single domain camelid antibodies (V_HHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast‐acting toxin and biothreat agent. The V1C7‐like V_HHs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin‐neutralizing activities. Using the X‐ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta‐based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1_R29G mutant was largely devoid of toxin‐neutralizing activity (TNA). However, the TNA of the V1C7_G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen‐deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.     

Production of Rhamnolipids by Pseudomonas chlororaphis, a Nonpathogenic Bacterium

Rhamnolipids, naturally occurring biosurfactants constructed of rhamnose sugar molecules and β-hydroxyalkanoic acids, have a wide range of potential commercial applications. In the course of a survey of 33 different bacterial isolates, we have identified, using a phenotypic assay for rhamnolipid production, a strain of the nonpathogenic bacterial species Pseudomonas chlororaphis that is capable of producing rhamnolipids. Rhamnolipid production by P. chlororaphis was achieved by growth at room temperature in static cultures of a mineral salts medium containing 2% glucose. We obtained yields of roughly 1 g/liter of rhamnolipids, an amount comparable to the production levels reported in Pseudomonas aeruginosa grown with glucose as the carbon source. The rhamnolipids produced by P. chlororaphis appear to be exclusively the mono-rhamnolipid form. The most prevalent molecular species had one monounsaturated hydroxy fatty acid of 12 carbons and one saturated hydroxy fatty acid of 10 carbons. P. chlororaphis, a nonpathogenic saprophyte of the soil, is currently employed as a biocontrol agent against certain types of plant fungal diseases. The pathogenic nature of all bacteria previously known to produce rhamnolipids has been a major obstacle to commercial production of rhamnolipids. The use of P. chlororaphis therefore greatly simplifies this matter by removing the need for containment systems and stringent separation processes in the production of rhamnolipids.     

Conversion of phytosterols into androstenedione by Mycobacterium neoaurum

Optimum conditions for transformation of phytosterols by Mycobacterium neoaururm, required for selective cleavage of the lateral chain into androstenedione, were shown to differ from the known conditions of animal sterol (cholesterol) transformation. Complete conversion of phytosterols into androstenedione at a substrate load of no less than 20 g/l was achieved on increasing the amount of the inoculum and the concentration of glucose (by 2 and 4 times, respectively, relative to cholesterol) and performing the fermentation under conditions of turbulent mixing. Under these conditions, both the rate of the transformation and the yield of the reaction product were high, due to the saturation of the culture liquid with hydrocarbonate. Data from the literature show that this ion is involved in cleavage of the branched lateral chain at carbon in position 24.     

Pharmacokinetics and Pharmacodynamic Effects of Flunixin after Intravenous,Intramuscular and Oral Administration to Dairy Goats

The pharmacokinetics and the prostaglandin (PG) synthesis inhibiting effect of flunixin were determined in 6 Norwegian dairy goats. The dose was 2.2 mg/kg body weight administered by intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) routes using a cross-over design. Plasma flunixin content was analysed by use of liquid chromatography and the PG synthesis was evaluated by measuring plasma 15-ketodihydro-PGF_2α by a radioimmuno-assay. Results are presented as median (range). The elimination half-lives (t_1/2·λ) were 3.6 (2.0–5.0), 3.4 (2.6–6.8) and 4.3 (3.4–6.1) h for i.v., i.m. and p.o. administration, respectively. Volume of distribution at steady state (Vd_ss) was 0.35 (0.23–0.41) L/kg and clearance (CL), 110 (60–160) mL/h/kg. The plasma concentrations after oral administration showed a double-peak phenomenon with the two peaks occurring at 0.37 (0.25–1) and 3.5 (2.5–5.0) h, respectively. Both peaks were in the same order of magnitude. Bioavailability was 79 (53–112) and 58 (35%–120)% for i.m. and p.o. administration, respectively. 15-Ketodihydro-PGF_2α plasma concentrations decreased after flunixin administration independent of the route of administration.     

The scaling of reproductive variability in trees

Seed output in perennial plant populations is temporally variable and often synchronous over large regions. The similarly complex spatiotemporal dynamics of animal populations have been characterized by the power‐law scaling of the variance in population numbers with mean abundance. Here we show that a large compilation of published reproductive time series exhibits largely invariant mean–variance scaling properties across both angiosperm and conifer tree species. A simple model of seed production in tree stands shows that observed values of the scaling exponent reflect very general aspects of plant ecology and life history as well as the temporal dynamics of seed production. Together, these results suggest that the continuum of reproductive variability and synchrony observed in trees may reflect the influence of a common set of ecological processes.     

Oligomeric Yeast Frataxin Drives Assembly of Core Machinery for Mitochondrial Iron-Sulfur Cluster Synthesis

Mitochondrial biosynthesis of iron-sulfur clusters (ISCs) is a vital process involving the delivery of elemental iron and sulfur to a scaffold protein via molecular interactions that are still poorly defined. Analysis of highly conserved components of the yeast ISC assembly machinery shows that the iron-chaperone, Yfh1, and the sulfur-donor complex, Nfs1-Isd11, directly bind to each other. This interaction is mediated by direct Yfh1-Isd11 contacts. Moreover, both Yfh1 and Nfs1-Isd11 can directly bind to the scaffold, Isu1. Binding of Yfh1 to Nfs1-Isd11 or Isu1 requires oligomerization of Yfh1 and can occur in an iron-independent manner. However, more stable contacts are formed when Yfh1 oligomerization is normally coupled with the binding and oxidation of Fe²⁺. Our observations challenge the view that iron delivery for ISC synthesis is mediated by Fe²⁺-loaded monomeric Yfh1. Rather, we find that the iron oxidation-driven oligomerization of Yfh1 promotes the assembly of stable multicomponent complexes in which the iron donor and the sulfur donor simultaneously interact with each other as well as with the scaffold. Moreover, the ability to store ferric iron enables oligomeric Yfh1 to adjust iron release depending on the presence of Isu1 and the availability of elemental sulfur and reducing equivalents. In contrast, the use of anaerobic conditions that prevent Yfh1 oligomerization results in inhibition of ISC assembly on Isu1. These findings suggest that iron-dependent oligomerization is a mechanism by which the iron donor promotes assembly of the core machinery for mitochondrial ISC synthesis.ISC³ biosynthesis is an essential function that eukaryotic cells initiate in mitochondria and probably other cellular compartments using three core components: a sulfur donor, an iron donor, and an ISC assembly scaffold (1, 2). In yeast mitochondria, the cysteine-desulfurase, Nfs1, and the iron-chaperone, Yfh1, are believed to provide sulfur and iron, respectively, for ISC assembly on the Isu1 scaffold (1), whereas the Nfs1-binding protein, Isd11, has been shown to stabilize Nfs1 (3). These components are highly conserved and the human orthologues of Yfh1 (frataxin), Isu1 (ISCU), and Isd11 (ISD11) are implicated in the etiology of severe disorders including Friedreich ataxia and mitochondrial myopathy (4).Previous studies have underscored the complexity of the interactions among eukaryotic ISC assembly components as well as their metal dependence. Supplementation of mitochondrial lysates with Fe²⁺ under aerobic conditions led to co-isolation of Yfh1 and Isu1 along with Nfs1 and Isd11 by pulldown or immunoprecipitation assays (5–7). Furthermore, aerobic preincubation of histidine-tagged Yfh1 monomer with Fe²⁺ enabled Isu1 to be pulled down by Yfh1 in the absence of other proteins (5). These studies have led to the current view that iron delivery for yeast ISC synthesis involves direct contacts between iron-loaded monomeric Yfh1 and Isu1 (5–7). Although Yfh1 oligomerization is normally coupled with iron binding, oxidation, and storage (5, 8), the possibility that Isu1 might also interact with oligomeric Yfh1 has remained largely unexplored.Similar to Yfh1, human frataxin was found to interact with multiple ISC assembly components in human cells; however, in this case immunoprecipitation data suggested that frataxin binds to ISCU indirectly, via nickel-dependent contacts with ISD11 (9). Whether direct interactions occur between Yfh1 and Isd11 has not yet been examined.While previous studies focused primarily on Yfh1-Isu1 and frataxin-ISD11 interactions, it is likely that the coordinate delivery of potentially toxic sulfur and iron to Isu1/ISCU involves multiple close interactions whereby the sulfur donor and the iron donor simultaneously interact with each other and with the ISC scaffold, as proposed for prokaryotic ISC assembly (10). However, it is currently unknown whether monomeric Yfh1/frataxin may form direct contacts with more than one partner, and the structure of the eukaryotic ISC assembly machinery is completely undefined. We show that iron oxidation-dependent oligomerization enables Yfh1 to have simultaneous direct interactions with Nfs1-Isd11 and Isu1. Our data provide insights about the sequence of events and the molecular architecture required for the initial step in mitochondrial ISC assembly.     

Isolation of highly enriched apical plasma membranes of the placental syncytiotrophoblast

The human placenta is a complex organ whose proper function is crucial for the development of the fetus. The placenta contains within its structure elements of the maternal and fetal circulatory systems. The interface with maternal blood is the lining of the placenta, that is a unique compartment known as the syncytiotrophoblast. This large syncytial structure is a single cell layer in thickness, and the apical plasma membrane of the syncytiotrophoblast interacts directly with maternal blood. Relatively little is known about the proteins that reside in this unique plasma membrane or how they may change in various placental diseases. Our goal was to develop methods for isolating highly enriched preparations of this apical plasma membrane compatible with high-quality proteomics analysis and herein describe the properties of these isolated membranes.     

Different Poses for Ligand and Chaperone in Inhibitor-Bound Hsp90 and GRP94: Implications for Paralog-Specific Drug Design

Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian Hsp90 paralogs, which are cytoplasmic Hsp90α and β, endoplasmic reticulum GRP94, and mitochondrial Trap-1. Given that each of the Hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one Hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each Hsp90 paralog. Here, we present side-by-side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-Hsp90 inhibitors geldanamycin (Gdm) and radamide. These structures reveal paralog-specific differences in the Hsp90 and GRP94 conformations in response to Gdm binding. We also report significant variation in the pose and disparate binding affinities for the Gdm-radicicol chimera radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors.