Principles and pitfalls in designing site-directed peptide ligands

An immunoglobulin light chain dimer with a large generic binding cavity was used as a host molecule for designing a series of peptide guest ligands. In a screening procedure peptides coupled to solid supports were systematically tested for binding activity by enzyme linked immunosorbent assays (ELISA). Key members of the binding series were synthesized in milligram quantities and diffused into crystals of the host molecule for X-ray analyses. These peptides were incrementally increased in size and affinity until they nearly filled the cavity. Progressive changes in binding patterns were mapped by comparisons of crystallo-graphically refined structures of 14 peptide–protein complexes at 2.7 Å resolution. These comparisons led to guidelines for ligand design and also suggested ways to modify previously established binding patterns. By manipulating equilibria involving histidine, for example, it was possible to abolish one important intramolecular interaction of the bound ligand and substitute another. These events triggered a change inconformation of the ligand from a compact to an extended form and a comprehensive change in the mode of binding to the protein. In dipeptides of histidine and proline, protonation of both imidazolium nitrogen atoms was used to program anend-to-end reversal of the direction in which the ligand was inserted into the binding cavity. Peptides cocrystallized with proteins produced complexes somewhat different in structure from those in which ligandswere diffused into preexisting crystals. In sucha large and malleable cavity, space utilization was thus different when a ligand was introduced before the imposition of crystal packing restraints. © 1993 Wiley-Liss, Inc.     

Morphological indices and otolith microstructure of Atlantic croaker,Micropogonias undulatus,as indicators of habitat quality along an estuarine pollution gradient

Synopsis Atlantic croaker, Micropogonias undulatus, sampled from a transect along a pollution gradient show a trend of declining growth and physical condition. This trend is apparent in the mean size of 0-group croaker, in their recent growth rate measured by marginal otolith increment widths, in longer term growth rate as indicated by relative otolith weights, and in general physical condition as measured by an index of condition of the caudal fin. We suggest that these measures are indicators of stress associated with environmental conditions. Because croaker from different positions along the pollution gradient were distinguishable, it appears that they remain for extended periods within areas of degraded environmental quality.     

Dual miRNA Targeting Restricts Host Range and Attenuates Neurovirulence of Flaviviruses

Mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. Vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. Some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially lead to a vaccine virus reversion back to wild-type in nature, followed by introduction and dissemination of potentially dangerous viral strains into new geographic locations. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3’NCR and/or ORF region resulted in selective restriction of dengue type 4 virus (DEN4) replication in mosquito cell lines and adult Aedes mosquitos. Moreover a combined targeting of DEN4 genome with mosquito-specific and vertebrate CNS-specific mir-124 miRNA can silence viral replication in two evolutionally distant biological systems: mosquitoes and mouse brains. Thus, this approach can reinforce the safety of newly developed or existing vaccines for use in humans and could provide an additional level of biosafety for laboratories using viruses with altered pathogenic or transmissibility characteristics.     

Severed intestine in channel catfish

Six cases of severed intestines in farm-raised channel catfish were examined at fish disease diagnostic laboratories in Mississippi and Alabama. This condition has not been reported previously in fish. Affected fish had a 4-7-cm-long intestinal section (hyperemic where it was severed) attached to the stomach. The remainder of the intestine was completely missing in all six cases except for a 1-1.5-cm section of intestine prolapsed from the anus in fish from three of the cases. Ischemia, autodigestion following intussusception, or intestinal epithelium degradation are suggested as possible etiologies for this condition.     

A model for the formation and interconversion of protein A-immunoglobulin G soluble complexes

We present a model for the formation and interconversion of the soluble complexes formed by reacting staphylococcal protein A (SpA) with rabbit immunoglobulin G (IgG) antibodies. The basic elements of the model are developed from reported hydrodynamic and electron microscopic studies of these complexes (see accompanying companion paper), together with established structural and binding properties of IgG and SpA. The model includes specific symmetry and binding requirements for IgG-SpA combination, and a steric constraint between neighboring IgG molecules. We discuss how such a constraint could influence the assembly and distribution of equilibrium complexes. After formulating a convenient symbolism for representing IgG-SpA complexes, the suggested model is used to construct plausible structures for the four predominant complexes observed in moderate SpA excess. Distributions of these stable complexes at different IgG:SpA ratios, together with LeChatelier's principle and a straightforward thermodynamic derivation, are used to predict likely arrangements of equilibrium structures. Also, a scale model of the unique IgG4-SpA2 complex formed in IgG excess is constructed from reported x-ray diffraction and amino acid sequence data. An intuitive thermodynamic argument is used to show that the suggested steric constraint could cause the rather unprecedented reversible transformation of the four 7 to 15S complexes into the unique 17S complex. A computer simulation is used to predict equilibrium concentrations of the various proposed complexes at different IgG:SpA ratios. In support of the suggested structures, the calculated thermodynamic distributions agree surprisingly well with those measured with the ultracentrifuge. We point out how the proposed arrangements of the complexes, and in particular the 17S complex, can account for many of their novel properties, such as antigen-induced conformational changes. Reported differences in complement activation and precipitate formation by SpA complexes formed with antibodies from various species are also discussed with regard to possible differences in structural arrangements of the complexes.