Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae

BACKGROUND: Porins are channel-forming membrane proteins that confer solute permeability to the outer membrane of Gram-negative bacteria. In Escherichia coli, major nonspecific porins are matrix porin (OmpF) and osmoporin (OmpC), which show high sequence homology. In response to high osmolarity of the medium, OmpC is expressed at the expense of OmpF porin. Here, we study osmoporin of the pathogenic Klebsiella pneumoniae (OmpK36), which shares 87% sequence identity with E. coliOmpC in an attempt to establish why osmoporin is best suited to function at high osmotic pressure. RESULTS: The crystal structure of OmpK36 has been determined to a resolution of 3.2 A by molecular replacement with the model of OmpF. The structure of OmpK36 closely resembles that of the search model. The homotrimeric structure is composed of three hollow 16-stranded antiparallel beta barrels, each delimiting a separate pore. Most insertions and deletions with respect to OmpF are found in the loops that protrude towards the cell exterior. A characteristic ten-residue insertion in loop 4 contributes to the subunit interface. At the pore constriction, the replacement of an alanine by a tyrosine residue does not alter the pore profile of OmpK36 in comparison with OmpF because of the different course of the mainchain. Functionally, as characterized in lipid bilayers and liposomes, OmpK36 resembles OmpC with decreased conductance and increased cation selectivity in comparison with OmpF. CONCLUSIONS: The osmoporin structure suggests that not an altered pore size but an increase in charge density is the basis for the distinct physico-chemical properties of this porin that are relevant for its preferential expression at high osmotic strength.     

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins.     

Botulinum neurotoxins C, E and F bind gangliosides via a conserved binding site prior to stimulation-dependent uptake with botulinum neurotoxin F utilising the three isoforms of SV2 as second receptor

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion.     

Coevolution of the glucose dehydrogenase gene and the ejaculatory duct in the genus Drosophila

The glucose dehydrogenase gene (Gld) in Drosophila melanogaster exhibits a unique spatial and temporal pattern of expression. GLD expression switches from a non-sex-limited state at the pupal stage to a male-limited state at the adult stage. At the adult stage, the enzyme is restricted to the ejaculatory duct. Within the genus Drosophila, the ejaculatory duct has undergone a simple morphological divergence. In order to determine whether correlated changes in GLD expression had occurred, GLD activity during the pupal and adult stages was determined for several Drosophila species. It was found that virtually all of the species exhibit pupal GLD activity, whereas only those species with an expanded ejaculatory duct express male-limited GLD. The results of interspecific genital imaginal disc transplantation experiments indicate that the expanded morphology and GLD expression do not require any species- or sex-specific diffusible factors. An apparent regulatory polymorphism exists within the D. takahashii species with respect to male-limited GLD expression.      

A report on methods to reduce,refine and replace animal testing in industrial toxicology laboratories

The Committee to Promote Principles of Reduction, Refinement and Replacement of Animal Testing in Industrial Toxicology Laboratories was established in 1987 to work toward industrywide improvements in laboratory animal testing methods. The committee's goals are to gather information about effective nonanimal testing techniques and other methods of conserving and improving the care of laboratory animals, to work toward the systematic validation of nonanimal alternatives, and to disseminate useful information about progressive programs and policies throughout the industrial toxicology community. This is the first in a continuing series of reports the committee plans to produce as part of an ongoing program to promote communication among industrial toxicologists about successful methods of reducing, refining and replacing animal testing. Here are some of the report's major findings: (1) Animal care and use committees charged with the oversight of laboratory animal use are a universal practice at the companies surveyed. (2) Significant reductions in the number of animals used for acute toxicity testing have taken place at all the companies during the last 5- to 10-year period. (3) Structure-activity relationships (predicting a test compound's properties based on the known properties of familiar chemicals with similar structures) are widely used to minimize, but not replace, the use of animals. (4) Tissue and organ culture systems are being used with increasing frequency for screening and mechanistic studies, but are not completely replacing animal evaluations as a final step. (5) There is a pressing need for the systematic and scientifically sound validation of nonanimal alternative techniques to reduce the use of animals in toxicology testing while satisfying requirements for the protection of public safety.     

2,6-Naphthyridines as potent and selective inhibitors of the novel protein kinase C isozymes

The present study describes a novel series of ATP-competitive PKC inhibitors based on the 2,6-naphthyridine template. Example compounds potently inhibit the novel Protein Kinase C (PKC) isotypes δ, ε, η, θ (in particular PKCε/η, and display a 10–100-fold selectivity over the classical PKC isotypes. The prototype compound 11 was found to inhibit PKCθ-dependent pathways in vitro and in vivo. In vitro, a-CD3/a-CD28-induced lymphocyte proliferation could be effectively blocked in 10% rat whole blood. In mice, 11 dose-dependently inhibited Staphylococcus aureus enterotoxin B-triggered IL-2 serum levels after oral dosing.