Generation of antiserum to specific epitopes

The ability to prevent disease by immunization with subunit vaccines that incorporate specific epitopes was demonstrated by DiMarchi et al. (1), who used a synthetic peptide to protect cattle against foot-and-mouth disease. However, generation of antibody to peptide antigens is often difficult owing to the small molecular mass and limited chemical complexity. We tested the hypothesis that recombinant DNA and synthetic peptide techniques would make it possible to stimulate vigorous immune responses to specific epitopes of an outer membrane protein ofNeisseria gonorrhoeae. The MtrC AP1 sequence from the invariant mtrC gonococcal lipoprotein was genetically fused to maltose binding protein. The resultant fusion protein was used as the primary immunogen to stimulate MtrC AP1-specific antiserum. To enhance antibody production specific to MtrC AP1, boosting immunizations were performed with synthetic MtrC AP1 sequence contained in a multiple antigenic peptide system immunogen. The MtrC AP1-specific antiserum strongly recognized the MtrC protein on Western blots and appeared to bind native MtrC proteinin situ. The generation of antibody in this fashion provides the technology to produce antibody to defined epitopes of any protein, including those found in the gonococcal outer membrane. The ability of those antibodies to inhibit bacterial growth or to activate complement protein can then be tested.     

The octamer motif in immunoglobulin genes: extraction of structural constraints from two-dimensional NMR studies.

Phase-sensitive two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlated (2QF-COSY) spectra were recorded at 500 MHz for the DNA duplex d(CATTTGCATC).d(GATGCAAATG), which contains the octamer element of immunoglobulin genes. Exchangeable and nonexchangeable proton resonances including those of the H5' and H5" protons were assigned. Overall, the decamer duplex adopts a B-type DNA conformation. Scalar coupling constants for the sugar protons were determined by quantitative simulations of 2QF-COSY cross-peaks. These couplings are consistent with a two-state dynamic equilibrium between a minor N- and a major S-type conformer for all residues. The pseudorotation phase angle P of the major conformer is in the range 117-135 degrees for nonterminal pyrimidine nucleotides and 153-162 degrees for nonterminal purine nucleotides. Except for the terminal residues, the minor conformer comprises less than 25% of the population. Distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities with the MARDIGRAS algorithm confirm the dependence of the sugar pucker on pyrimidine and purine bases. Averaging by fast local motions has at most small effects on the NOE-derived interproton distances.     

Spectrophotometric and NMR analysis of the interaction of fluorinated mono- and bifunctional intercalators with DNA, poly(dA-dT), and poly(dG-dC)

We have synthesized and investigated the DNA binding properties of three fluorinated acridine derivatives—a monomer (I), a short dimer (II) and a long dimer (III). Only III has a sufficiently long chain bridging the two acridine nuclei to permit binding by bisintercalation. Analysis of the equilibrium and kinetic binding properties of these compounds to poly(dA-dT) demonstrates that they behave very similarly to their unfluorinated parent compounds. Helix extension, as determined by viscosity measurements, shows that both compounds I and II bind by monointercalation while III binds by bisintercalation. These results are confirmed by ¹⁹F-nmr analysis, which indicates, in particular, that the two chromophores of III share the same molecular environment as that of I in the presence of either calf thymus DNA or poly(dA-dT). Negative nuclear Overhauser effects in the presence of DNA indicate tight binding such that the motion of the ligands is governed by the polynucleotide dynamics. Optical titrations establish that in 4M NaCl, both I and III bind to calf thymus DNA, but no binding was observed with poly(dG-dC). This result is in contrast to those for dimers of ethidium, which show substantial binding to polynucleotides under high salt conditions. Nuclear magnetic resonance experiments, however, carried out at considerably higher concentrations, show that compound I does indeed bind to poly(dG-dC) under these high salt conditions, albeit weakly, and leads to a conversion of the polynucleotide from a left-handed to a right-handed conformation.     

Long range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains

The dynein motor domain consists of a ring of six AAA domains with a protruding microtubule-binding stalk and a C-terminal domain of unknown function. To understand how conformational information is communicated within this complex structure, we produced a series of recombinant and proteolytic rat motor domain fragments, which we analyzed enzymatically. A recombinant 210-kDa half-motor domain fragment surprisingly exhibited a 6-fold higher steady state ATPase activity than a 380-kDa complete motor domain fragment. The increased ATPase activity was associated with a complete loss of sensitivity to inhibition by vanadate and an approximately 100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated approximately 1000-fold by microtubule binding to the 380-kDa motor domain. Both the half-motor and full motor domain fragments were remarkably resistant to tryptic proteolysis, exhibiting either two or three major cleavage sites. Cleavage near the C terminus of the 380-kDa motor domain released a 32-kDa fragment and abolished sensitivity to vanadate. Cleavage at this site was insensitive to ATP or 5'-adenylyl-beta,gamma-imidodiphosphate but was blocked by ADP-AlF3 or ADP-vanadate. Based on these data, we proposed a model for long range allosteric control of product release at AAA1 and AAA3 through the microtubule-binding stalk and the C-terminal domain, the latter of which may interact with AAA1 to close the motor domain ring in a cross-bridge cycle-dependent manner.     

Meta‐barcoding insights into the spatial and temporal dietary patterns of the threatened Asian Great Bustard (Otis tarda dybowskii) with potential implications for diverging migratory strategies

Food resources are often not sufficient to satisfy the nutritional and energetic requirements during winter conditions at high latitudes. Dietary analysis is a prerequisite to fully understanding the feeding ecology of a species and the nature of trophic interactions. Previous dietary studies of Asian Great Bustard (Otis tarda dybowskii) relied on behavioral observations, resulting in categorization of diet limited to broad taxonomic groupings. Here, we applied a high‐throughput sequencing meta‐barcoding approach to quantify the diet of resident and migratory Asian Great Bustard in three wintering sites during early winter and late winter. We detected 57 unique plant taxa in the bustard diet, among which 15 species were confirmed by a local plant database we generated. Both agricultural and natural foods were detected, indicating a relatively broad dietary niche. Spatiotemporal dietary changes were discovered, revealing diet differences among wintering sites and a general shift toward lower plant diversity later in winter. For the nonmigratory population, we detected a significantly more diverse array of plant species in their diet. We hypothesize that dietary variation between resident and migratory populations could be involved in the recent transition to partial migration in this species, although climate change can not be excluded. Collectively, these results support protecting unharvested grain fields and naturally unplowed lands to help conserve and promote population growth of Asian Great Bustard.     

Neisseria gonorrhoeae Modulates Iron-Limiting Innate Immune Defenses in Macrophages

Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted infection termed gonorrhea. The gonococcus can survive extracellularly and intracellularly, but in both environments the bacteria must acquire iron from host proteins for survival. However, upon infection the host uses a defensive response by limiting the bioavailability of iron by a number of mechanisms including the enhanced expression of hepcidin, the master iron-regulating hormone, which reduces iron uptake from the gut and retains iron in macrophages. The host also secretes the antibacterial protein NGAL, which sequesters bacterial siderophores and therefore inhibits bacterial growth. To learn whether intracellular gonococci can subvert this defensive response, we examined expression of host genes that encode proteins involved in modulating levels of intracellular iron. We found that N. gonorrhoeae can survive in association (tightly adherent and intracellular) with monocytes and macrophages and upregulates a panel of its iron-responsive genes in this environment. We also found that gonococcal infection of human monocytes or murine macrophages resulted in the upregulation of hepcidin, NGAL, and NRAMP1 as well as downregulation of the expression of the gene encoding the short chain 3-hydroxybutyrate dehydrogenase (BDH2); BDH2 catalyzes the production of the mammalian siderophore 2,5-DHBA involved in chelating and detoxifying iron. Based on these findings, we propose that N. gonorrhoeae can subvert the iron-limiting innate immune defenses to facilitate iron acquisition and intracellular survival.