The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

In vitro evaluation of native entomopathogenic fungi and neem (Azadiractha indica) extracts on Spodoptera frugiperda

The control of Spodoptera frugiperda is based on synthetic insecticides, so some alternatives are the use of entomopathogenic fungi (EF) and neem extract. The objective of the study was to evaluate in vitro effectiveness of native EF and neem extracts on S. frugiperda larvae. Six EF were identified by DNA sequencing of ITS regions from three EF (Fusarium solani, Metarrhizium robertsii, Nigrospora spherica and Penicillium citrinum). They were evaluated in concentrations of 1 × 10⁸ spores/ mL. In addition, a second bioassay was carried out evaluating only F. solani, M. robertsii and N. sphaerica and the addition of vegetable oil. On the other hand, extraction of secondary metabolites from neem seed (Azadirachta indica) was carried out by performing, mass (g) and solvent volume (mL ethanol and water) combinations, which were subjected to microwaves and ultrasound. Subsequently, these extracts were evaluated in concentrations of 3%, 4% and 5%. A survival analysis was performed for each of the bioassays. With respect to the results of the first bioassay, F. solani obtained a probability of survival of 0.476 on the seventh day, while in the second bioassay, M. robertsii obtained 0.488 survival probability. This suggests that the expected percentage of larvae that stay alive on the sixth day is 48.8%. However, in the evaluation of the neem extract the combination 1:12/70% to 4% caused 84% mortality of larvae. The use of native HE and neem extracts has potential for the control of S. frugiperda.     

Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H

Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.     

Cytosolic delivery of granzyme B by bacterial toxins: evidence that endosomal disruption, in addition to transmembrane pore formation, is an important function of perforin

Granule-mediated cell killing by cytotoxic lymphocytes requires the combined actions of a membranolytic protein, perforin, and granule-associated granzymes, but the mechanism by which they jointly kill cells is poorly understood. We have tested a series of membrane-disruptive agents including bacterial pore-forming toxins and hemolytic complement for their ability to replace perforin in facilitating granzyme B-mediated cell death. As with perforin, low concentrations of streptolysin O and pneumolysin (causing <10% (51)Cr release) permitted granzyme B-dependent apoptosis of Jurkat and Yac-1 cells, but staphylococcal alpha-toxin and complement were ineffective, regardless of concentration. The ensuing nuclear apoptotic damage was caspase dependent and included cleavage of poly(ADP-ribose) polymerase, suggesting a mode of action similar to that of perforin. The plasma membrane lesions formed at low dose by perforin, pneumolysin, and streptolysin did not permit diffusion of fluorescein-labeled proteins as small as 8 kDa into the cell, indicating that large membrane defects are not necessary for granzymes (32 to 65 kDa) to enter the cytosol and induce apoptosis. The endosomolytic toxin, listeriolysin O, also effected granzyme B-mediated cell death at concentrations which produced no appreciable cell membrane damage. Cells pretreated with inhibitors of endosomal trafficking such as brefeldin A took up granzyme B normally but demonstrated seriously impaired nuclear targeting of granzyme B when perforin was also added, indicating that an important role of perforin is to disrupt vesicular protein trafficking. Surprisingly, cells exposed to granzyme B with perforin concentrations that produced nearly maximal (51)Cr release (1,600 U/ml) also underwent apoptosis despite excluding a 8-kDa fluorescein-labeled protein marker. Only at concentrations of >4,000 U/ml were perforin pores demonstrably large enough to account for transmembrane diffusion of granzyme B. We conclude that pore formation may allow granzyme B direct cytosolic access only when perforin is delivered at very high concentrations, while perforin's ability to disrupt endosomal trafficking may be crucial when it is present at lower concentrations or in killing cells that efficiently repair perforin pores.     

Fas-ligand-mediated lysis of erbB-2-expressing tumour cells by redirected cytotoxic T lymphocytes

A chimeric receptor, consisting of the single-chain variable (scFv) domains of an anti-erbB-2 mAb linked via a CD8 membrane-proximal hinge to the Fc receptor γ chain, was expressed in the mouse cytotoxic T lymphocyte (CTL) hybridoma cell line, MD45. This cell line was grafted with the additional specificity to recognise and bind erbB-2-expressing breast carcinoma target cells T47D, MCF-7 and BT-20 in a non-MHC-restricted manner. Tumour cell lysis was antigen-specific since erbB-2-negative tumours were insensitive to lysis by MD45-scFv-anti-erbB-2-γ clones, and lysis of erbB-2⁺ tumour targets was inhibited in the presence of an anti-erbB-2 mAb. Furthermore, target cell death correlated with the level of chimeric receptor expression on the effector MD45 subclones. Redirected MD45 CTL utilised Fas ligand to induce target cell death since soluble Fas-Fc fusion protein completely inhibited cytolysis. The sensitivity of tumour target cells to Fas ligand was further enhanced by treating them with interferon-γ, a regulator of Fas and downstream signalling components of the Fas pathway. Overall, this study has demonstrated the requirement for successful activation of Fas ligand function in conjunction with cytokine treatment for effective lysis of breast carcinoma target cells mediated by redirected CTL. Received: 23 July 1998 / Accepted: 5 October 1998     

Domain motions and quaternary packing of phosphofructokinase-2 from Escherichia coli studied by small angle x-ray scattering and homology modeling

The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme. When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure. Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level. We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures. The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex. Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer. We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure. Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure. Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.     

Functional significance of the perforin/granzyme cell death pathway

Perforin/granzyme-induced apoptosis is the main pathway used by cytotoxic lymphocytes to eliminate virus-infected or transformed cells. Studies in gene-disrupted mice indicate that perforin is vital for cytotoxic effector function; it has an indispensable, but undefined, role in granzyme-mediated apoptosis. Despite its vital importance, the molecular and cellular functions of perforin and the basis of perforin and granzyme synergy remain poorly understood. The purpose of this review is to evaluate critically recent findings on cytotoxic granule-mediated cell death and to assess the functional significance of postulated cell-death pathways in appropriate pathophysiological contexts, including virus infection and susceptibility to experimental or spontaneous tumorigenesis.     

Peripheral benzodiazepine receptor ligand-melphalan conjugates for potential selective drug delivery to brain tumors

To gain insight into the strategy to target PBR ligand-drug conjugates to brain tumors, novel N-imidazopyridinacetyl-melphalan conjugates and the corresponding ethyl esters have been prepared and evaluated for their cytotoxicity in melphalan-sensitive human (SF126, SF188) and rat (RG-2) glioma cell lines. These conjugates exhibited PBR binding affinity with IC(50) values ranging from 57 and 2614 nM. By a computational approach it can be predicted that these conjugates possess significant brain penetration. The stability of the conjugates in 0.05 M phosphate buffer at pH 7.4 and, in some cases, in dilute human serum solution was determined. All the ethyl ester derivatives were stable in 0.05 M phosphate buffer at pH 7.4 and their half-lives exceeded 28 h. Conversely, under the same conditions, the corresponding acids were found to undergo a fast cleavage within a few minutes. HPLC-MS analysis of the mixture from degradation in buffer and physiological medium of the representative cases allowed the identification of their main degradation products. A plausible degradation pathway accounting for the available experimental data is presented.