Crystallization of porin using short chain phospholipids

A protein from outer membranes of Escherichia coli K-12, porin OmpF was crystallized either with short acyl chain phospolipids or with various detergents as amphiphiles. In dihexanoyl phosphatidylcholine, crystals greater than 0.3 mm in all dimensions were obtained that diffract X-rays beyond 3.5 A. Crystal morphology and unit cell dimensions were indistinguishable from those of detergent-grown crystals, indicating that the role of the phospholipids used is similar to those of many conventional detergents.     

Disruption of the viral polymerase complex assembly as a novel approach to attenuate influenza A virus

To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/β receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.     

Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme

The associated heavy (VH) and light (VL) chain variable domains (Fv) of the monoclonal anti-lysozyme antibody D1.3, secreted from Escherichia coli, have been crystallized in their antigen-bound and free forms. FvD1.3 gives tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with a = 90.6 A, c = 56.4 A. The FvD1.3-lysozyme complex crystallizes in space group C2, with a = 129.2 A, b = 60.8 A, c = 56.9 A and beta = 119.3 degrees. The crystals contain one molecule of Fv or of the Fv-lysozyme complex in their asymmetric units and diffract X-rays to high resolution, making them suitable for X-ray crystallographic studies.     

KCa2 channels activation prevents [Ca2+]i deregulation and reduces neuronal death following glutamate toxicity and cerebral ischemia

Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca²⁺]_i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca²⁺]_i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (K_Ca2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of K_Ca2.2 channels within 3 h after the onset of glutamate exposure. Activation of K_Ca2 channels preserved K_Ca2 expression and significantly reduced pathological increases in [Ca²⁺]_i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for K_Ca2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.     

Tetanus toxin light chain expression in Sertoli cells of transgenic mice causes alterations of the actin cytoskeleton and disrupts spermatogenesis.

Tetanus toxin is a powerful neurotoxin known to inhibit neurotransmitter release. The tetanus toxin light chain is a metalloprotease that cleaves some members of the synaptobrevin gene family with high specificity. Here, we report the expression of a synthetic gene encoding the tetanus toxin light chain in the seminiferous epithelium of transgenic mice. Spermatogenesis was severely impaired and mature spermatozoa were completely absent. Late spermatids exhibited pleomorphic shapes and acrosomal distortions. The number of Leydig cells was greatly increased. In situ hybridization analysis revealed that the toxin acts on Sertoli cells. Affected cells exhibited an aberrant distribution of actin filaments and many cells contained large vacuoles. Our results demonstrate that tetanus toxin is active in non-neuronal cells and suggest an important function for members of the synaptobrevin gene family during the late stages of spermatogenesis.     

Mechanism of Thiosulfate Oxidation in the SoxA Family of Cysteine-ligated Cytochromes

Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism.     

Delineation of several DR-restricted tetanus toxin T cell epitopes

We have characterized five human T cell clones specific for tetanus toxin. The combination of different techniques allowed us to precisely map two T cell epitopes within fragments 830-843 and 1273-1284 of tetanus toxin, as formally demonstrated by the use of corresponding synthetic peptides. The three other T cell clones were specific for regions 2-602, 604-742, and 865-1315 of tetanus toxin, respectively. The five T cell clones were shown to be restricted to HLA-DR Ag. Furthermore, the allele of HLA-DR utilized by the various epitopes has been determined. The use of HLA-DR-transfected L cells as APC directly demonstrated that two epitopes, one of which represented by fragment 1273-1284, were recognized in association with HLA-DRw52a. For the other three T cell epitopes, the data strongly suggested they were recognized in association with HLA-DR5. Finally, a sixth T cell clone was shown to be specific for tetanus toxoid, the vaccinal preparation of tetanus toxin, and not for other tetanus toxin fragments. This indicated that immunization with tetanus toxoid probably elicits a T cell response directed only in part against native tetanus toxin.     

Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons

Vinpocetine (ethyl apovincaminate), a synthetic derivative of the Vinca minor alkaloid vincamine, is widely used for the treatment of cerebrovascular-related diseases. One of the proposed mechanisms underlying its action is to protect against the cytotoxic effects of glutamate overexposure. Glutamate excitotoxicity leads to the disregulation of mitochondrial function and neuronal metabolism. As Vinpocetine has a binding affinity to the peripheral-type benzodiazepine receptor (PBR) involved in the mitochondrial transition pore complex, we investigated whether neuroprotection can be at least partially due to Vinpocetine’s effects on PBRs.Neuroprotective effects of PK11195 and Ro5-4864, two drugs with selective and high affinity to PBR, were compared to Vinpocetine in glutamate excitotoxicity assays on primary cortical neuronal cultures. Vinpocetine exerted a neuroprotective action in a 1–50 μM concentration range while PK11195 and Ro5-4864 were only slightly neuroprotective, especially in high (>25 μM) concentrations. Combined pretreatment of neuronal cultures with Vinpocetine and PK11195 or Ro5-4864 showed increased neuroprotection in a dose-dependent manner, indicating that the different drugs may have different targets. To test this hypothesis, mitochondrial membrane potential (MMP) of cultured neurons was measured by flow cytometry. 25 μM Vinpocetine reduced the decrease of mitochondrial inner membrane potential induced by glutamate exposure, but Ro5-4864 in itself was found to be more potent to block glutamate-evoked changes in MMP. Combination of Ro5-4864 and Vinpocetine treatment was found to be even more effective.In summary, the present results indicate that the neuroprotective action of vinpocetine in culture can not be explained by its effect on neuronal PBRs alone and that additional drug targets are involved.