Combining a noble gas with radiotherapy: glutamate receptor antagonist xenon may act as a radiosensitizer in glioblastoma.

BACKGROUND: Ionotropic glutamate receptors α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) modulate proliferation, invasion and radioresistance in glioblastoma (GB). Pharmacological targeting is difficult as many in vitro-effective agents are not suitable for in patient applications. We aimed to develop a method to test the well tolerated AMPAR- and NMDAR-antagonist xenon gas as a radiosensitizer in GB. METHODS: We designed a diffusion-based system to perform the colony formation assay (CFA), the radiobiological gold standard, under xenon exposure. Stable and reproducible gas atmosphere was validated with oxygen and carbon dioxide as tracer gases. After checking for AMPAR and NMDAR expression via immunofluorescence staining we performed the CFA with the glioblastoma cell lines U87 and U251 as well as the non-glioblastoma derived cell line HeLa. Xenon was applied after irradiation and additionally tested in combination with NMDAR antagonist memantine. RESULTS: The gas exposure system proved compatible with the CFA and resulted in a stable atmosphere of 50% xenon. Indications for the presence of glutamate receptor subunits were present in glioblastoma-derived and HeLa cells. Significantly reduced clonogenic survival by xenon was shown in U87 and U251 at irradiation doses of 4-8 Gy and 2, 6 and 8 Gy, respectively (p < 0.05). Clonogenic survival was further reduced by the addition of memantine, showing a significant effect at 2-8 Gy for both glioblastoma cell lines (p < 0.05). Xenon did not significantly reduce the surviving fraction of HeLa cells until a radiation dose of 8 Gy. CONCLUSION: The developed system allows for testing of gaseous agents with CFA. As a proof of concept, we have, for the first time, unveiled indications of radiosensitizing properties of xenon gas in glioblastoma.

Protein Kinase A Is Responsible for the Presynaptic Inhibition of Glycinergic and Glutamatergic Transmissions by Xenon in Rat Spinal Cord and Hippocampal CA3 Neurons.

The effects of a general anesthetic xenon (Xe) on spontaneous, miniature, electrically evoked synaptic transmissions were examined using the "synapse bouton preparation," with which we can clearly evaluate pure synaptic responses and accurately quantify pre- and postsynaptic transmissions. Glycinergic and glutamatergic transmissions were investigated in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xe presynaptically inhibited spontaneous glycinergic transmission, the effect of which was resistant to tetrodotoxin, Cd2+, extracellular Ca2+, thapsigargin (a selective sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor), SQ22536 (an adenylate cyclase inhibitor), 8-Br-cAMP (membrane-permeable cAMP analog), ZD7288 (an hyperpolarization-activated cyclic nucleotide-gated channel blocker), chelerythrine (a PKC inhibitor), and KN-93 (a CaMKII inhibitor) while being sensitive to PKA inhibitors (H-89, KT5720, and Rp-cAMPS). Moreover, Xe inhibited evoked glycinergic transmission, which was canceled by KT5720. Like glycinergic transmission, spontaneous and evoked glutamatergic transmissions were also inhibited by Xe in a KT5720-sensitive manner. Our results suggest that Xe decreases glycinergic and glutamatergic spontaneous and evoked transmissions at the presynaptic level in a PKA-dependent manner. These presynaptic responses are independent of Ca2+ dynamics. We conclude that PKA can be the main molecular target of Xe in the inhibitory effects on both inhibitory and excitatory neurotransmitter release. SIGNIFICANCE STATEMENT: Spontaneous and evoked glycinergic and glutamatergic transmissions were investigated using the whole-cell patch clamp technique in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xenon (Xe) significantly inhibited glycinergic and glutamatergic transmission presynaptically. As a signaling mechanism, protein kinase A was responsible for the inhibitory effects of Xe on both glycine and glutamate release. These results may help understand how Xe modulates neurotransmitter release and exerts its excellent anesthetic properties.

Structure, energetics, and spectroscopy of the K2+(X2Σ+g) interacting with the noble gas atoms Ar, Kr and Xe.

The structure, energetic, and spectroscopy properties of the ionic system K2+(X2Σ+g) interacting with the noble gas atoms Argon, Krypton and Xenon are studied. The computations are done by an accurate ab initio approach based on the pseudo-potential technique, Gaussian basis sets, parameterized l-dependent polarization potentials and an analytic potential form for the K+Ar, K+Kr and K+Xe interactions. These interactions are added via the CCSD(T) potential taken from literature and fitted applying the analytical expression of Tang and Toennies. The application of the pseudo-potential approach reduces the number of active electrons of each complex to only one electron. The potential energy surfaces are analyzed on a large range of the Jacobi coordinates, R and θ. By the general interpolation outline based on the RKHS (Reproducing Kernel Hilbert Space) procedure, we have reproduced for each complex from our ab initio results the two-dimensional contour plots of an analytical potential. To evaluate the stability of each complex, we have determined from the potential energy surfaces the equilibrium distance (Re), the well depth (De), the quantum energy (D0), the zero-point-energy (ZPE) and the ZPE%. The results showed that the linear configurations, where the noble gas atom connected to the K2+(X2Σ+g) system, are the more stable.

[Xenon post-conditioning protects against spinal cord ischemia-reperfusion injury in rats by downregulating mTOR pathway and inhibiting endoplasmic reticulum stress-induced neuronal apoptosis].

OBJECTIVE: The purpose of this study was to determine whether xenon post-conditioning affects mTOR signaling as well as endoplasmic reticulum stress (ERS)-apoptosis pathway in rats with spinal cord ischemia/reperfusion injury. METHODS: Fifty male rats were randomized equally into sham-operated group (Sham group), I/R model group (I/R group), I/R model+ xenon post-conditioning group (Xe group), I/R model+rapamycin (a mTOR signaling pathway inhibitor) treatment group (I/R+ Rapa group), and I/R model + xenon post- conditioning with rapamycin treatment group (Xe + Rapa group).. In the latter 4 groups, SCIRI was induced by clamping the abdominal aorta for 85 min followed by reperfusion for 4 h. Rapamycin (or vehicle) was administered by daily intraperitoneal injection (4 mg/kg) for 3 days before SCIRI, and xenon post-conditioning by inhalation of 1∶1 mixture of xenon and oxygen for 1 h at 1 h after initiation of reperfusion; the rats without xenon post-conditioning were given inhalation of nitrogen and oxygen (1∶ 1). After the reperfusion, motor function and histopathologic changes in the rats were examined. Western blotting and real-time PCR were used to detect the protein and mRNA expressions of GRP78, ATF6, IRE1α, PERK, mTOR, p-mTOR, Bax, Bcl-2 and caspase-3 in the spinal cord. RESULTS: The rats showed significantly lowered hind limb motor function following SCIRI (P < 0.01) with a decreased count of normal neurons, increased mRNA and protein expressions of GRP78, ATF6, IRE1α, PERK, and caspase-3, and elevated p-mTOR/mTOR ratio and Bax/Bcl-2 ratio (P < 0.01). Xenon post-conditioning significantly decreased the mRNA and protein levels of GRP78, ATF6, IRE1α, PERK and caspase-3 (P < 0.05 or 0.01) and reduced p-mTOR/mTOR and Bax/Bcl-2 ratios (P < 0.01) in rats with SCIRI; the mRNA contents and protein levels of GRP78 and ATF6 were significantly decreased in I/R+Rapa group (P < 0.01). Compared with those in Xe group, the rats in I/R+Rapa group and Xe+Rapa had significantly lowered BBB and Tarlov scores of the hind legs (P < 0.01), and caspase-3 protein level and Bax/Bcl-2 ratio were significantly lowered in Xe+Rapa group (P < 0.05 or 0.01). CONCLUSION: By inhibiting ERS and neuronal apoptosis, xenon post- conditioning may have protective effects against SCIRI in rats. The mTOR signaling pathway is partially involved in this process.

Pulmonary hyperpolarized (129) Xe morphometry for mapping xenon gas concentrations and alveolar oxygen partial pressure: Proof-of-concept demonstration in healthy and COPD subjects.

PURPOSE: Diffusion-weighted (DW) hyperpolarized (129) Xe morphometry magnetic resonance imaging (MRI) can be used to map regional differences in lung tissue micro-structure. We aimed to generate absolute xenon concentration ([Xe]) and alveolar oxygen partial pressure (pA O2 ) maps by extracting the unrestricted diffusion coefficient (D0 ) of xenon as a morphometric parameter. METHODS: In this proof-of-concept demonstration, morphometry was performed using multi b-value (0, 12, 20, 30 s/cm(2) ) DW hyperpolarized (129) Xe images obtained in four never-smokers and four COPD ex-smokers. Morphometric parameters and D0 maps were computed and the latter used to generate [Xe] and pA O2 maps. Xenon concentration phantoms estimating a range of values mimicking those observed in vivo were also investigated. RESULTS: Xenon D0 was significantly increased (P = 0.035) in COPD (0.14 ± 0.03 cm(2) /s) compared with never-smokers (0.12 ± 0.02 cm(2) /s). COPD ex-smokers also had significantly decreased [Xe] (COPD = 8 ± 7% versus never-smokers = 13 ± 8%, P = 0.012) and increased pA O2 (COPD = 18 ± 3% versus never-smokers = 15 ± 3%, P = 0.009) compared with never-smokers. Phantom measurements showed the expected dependence of D0 on [Xe] over the range of concentrations anticipated in vivo. CONCLUSION: DW hyperpolarized (129) Xe MRI morphometry can be used to simultaneously map [Xe] and pA O2 in addition to providing micro-structural biomarkers of emphysematous destruction in COPD. Phantom measurements of D0 ([Xe]) supported the hypotheses that differences in subjects may reflect differences in functional residual capacity.

Hyperpolarized (129)Xe spectra from C6 glioma cells implanted in rat brains.

Tumor cell density is dramatically different from normal tissue. Since the chemical shift of hyperpolarized (129)Xe reflects local cell structure, we hypothesized that the presence of tumor cells could potentially be determined from (129)Xe spectra. Spectra and washout decay rate from three rats implanted with C6 glioma cells were compared with eight control rats. No significant differences between normal and tumor spectra were observed. The decay time of the C6 rats (mean 13.5 ± 1.9 s) was not significantly different from normal rats (mean 11.7 ± 1.8 s). These results suggest that hyperpolarized Xe may not be a superior tracer for detection of tumor cells in the intact brain.

Structural plasticity of the phage P22 tail needle gp26 probed with xenon gas.

The tail needle, gp26, is a highly stable homo-trimeric fiber found in the tail apparatus of bacteriophage P22. In the mature virion, gp26 is responsible for plugging the DNA exit channel, and likely plays an important role in penetrating the host cell envelope. In this article, we have determined the 1.98 A resolution crystal structure of gp26 bound to xenon gas. The structure led us to identify a calcium and a chloride ion intimately bound at the interior of alpha-helical core, as well as seven small cavities occupied by xenon atoms. The two ions engage in buried polar interactions with gp26 side chains that provide specificity and register to gp26 helical core, thus enhancing its stability. Conversely, the distribution of xenon accessible cavities correlates well with the flexibility of the fiber observed in solution and in the crystal structure. We suggest that small internal cavities in gp26 between the helical core and the C-terminal tip allow for flexible swinging of the latter, without affecting the overall stability of the protein. The C-terminal tip may be important in scanning the bacterial surface in search of a cell-envelope penetration site, or for recognition of a yet unidentified receptor on the surface of the host.

Generation of noble-gas binding sites for crystallographic phasing using site-directed mutagenesis.

In recent years, the use of noble-gas complexes for the de novo phasing of protein structures has proven to be a useful alternative to selenomethionine and more traditional derivatives, largely owing to improvements in methods for incorporating noble gases within protein crystals. Advantages of noble-gas derivatives include a high degree of isomorphism when using xenon for multiple isomorphous replacement (MIR) and an easily accessible absorption edge when using krypton for multiwavelength anomalous dispersion (MAD) phasing. One problem with this approach is that not all proteins contain cavities which bind noble gases. Even in proteins which do bind noble gases, the resulting derivative may not be sufficient for phasing. Using T4 lysozyme as an example, it is illustrated how this limitation might be overcome by using 'large-to-small' mutations to introduce potential noble-gas binding sites. Wild-type T4 lysozyme contains a single xenon-binding site. By truncating leucine and phenylalanine residues to alanine, it is possible to generate additional noble-gas binding sites within the core of the protein. Combining rotating-anode data from xenon complexes of wild-type and mutant lysozymes yields MIR phases which compare favorably with those determined from a selenomethionine MAD experiment conducted at a synchrotron. Experience with T4 lysozyme suggests that a leucine-to-alanine substitution made at random in a protein of unknown structure has about a 30% chance of providing a useful derivative. This procedure holds promise for the determination of unknown protein structures, especially when selenomethionine-containing protein is not available or when access to a synchrotron is limited.

Structure of the soluble domain of a membrane-anchored thioredoxin-like protein from Bradyrhizobium japonicum reveals unusual properties.

TlpA is an unusual thioredoxin-like protein present in the nitrogen-fixing soil bacterium Bradyrhizobium japonicum. A hydrophobic N-terminal transmembrane domain anchors it to the cytoplasmic membrane, whereby the hydrophilic thioredoxin domain becomes exposed to the periplasmic space. There, TlpA catalyses an essential reaction, probably a reduction, in the biogenesis of cytochrome aa(3). The soluble thioredoxin domain (TlpA(sol)), devoid of the membrane anchor, was purified and crystallized. Oxidized TlpA(sol) crystallized as a non-covalent dimer in the space group P2(1)2(1)2(1). The X-ray structure analysis was carried out by isomorphous replacement using a xenon derivative. This resulted in a high-resolution (1.6 A) three-dimensional structure that displayed all of the features of a classical thioredoxin fold. A number of peculiar structural details were uncovered: (i) Only one of the two active-site-cysteine sulphurs (Cys72, the one closer to the N terminus) is exposed on the surface, making it the likely nucleophile for the reduction of target proteins. (ii) TlpA(sol) possesses a unique structural disulphide bond, formed between Cys10 and Cys155, which connects an unprecedented N-terminal alpha helix with a beta sheet near the C terminus. (iii) An insertion of about 25 amino acid residues, not found in the thioredoxin prototype of Escherichia coli, contributes only marginally to the thioredoxin fold, but forms an extra, surface-exposed alpha helix. This region plus another surface-exposed stretch (-Ile-Gly-Arg-Ala-), which is absent even in the closest TlpA relatives, might be considered as specificity determinants for the recognition of target proteins in the periplasm. The TlpA(sol) structure paves the way towards unraveling important structure-function relationships by rational mutagenesis.

Gas access to the active site of Ni-Fe hydrogenases probed by X-ray crystallography and molecular dynamics.

The 2.54 A resolution structure of Ni-Fe hydrogenase has revealed the existence of hydrophobic channels connecting the molecular surface to the active site. A crystallographic analysis of xenon binding together with molecular dynamics simulations of xenon and H2 diffusion in the enzyme interior suggest that these channels serve as pathways for gas access to the active site.

Discrepancy of xenon concentrations between end-tidal and blood collection methods in xenon-enhanced computed tomographic measurements of cerebral blood flow.

Using xenon-enhanced computed tomography for the study of cerebral blood flow, simultaneous measurements of end-tidal and arterial blood xenon concentrations using the blood collection method were performed to investigate the validity of substituting the end-tidal for the arterial blood xenon concentration. Simultaneous measurement by both methods was performed 68 times in 27 patients. There was no statistical correlation between the arterial blood accumulation rate constant obtained by arterial blood and end-tidal samples, nor between the arterial blood saturation value obtained by the two methods, even when correction was made for age. In brain tissue, all parameters calculated using the end-tidal concentration were lower than those using arterial blood. We therefore suggest that cerebral blood flow values calculated using end-tidal xenon concentration are useful only for qualitative cerebral blood flow mapping, and not applicable to absolute values of cerebral blood flow.

Application of xenon gas to clinical retinal detachment.

Xenon gas was used as an intravitreal tamponade in four cases of retinal detachment. The rapid disappearance of the gas could be predicted by experiments with an animal model, and there was no notable hypotensive phase. The brief presence of the gas was sufficient to press out retinal folds and effect reattachment. The uveal response was less than with air or with the longer-lasting gases. The time the patient had to be in a prone or bent position was reduced to a few hours.

Stochastic description of inert gas exchange.

Data from experiments measuring the rate of uptake and elimination of nonmetabolic gas in living organs are usually analyzed by several exponential time constants or their equivalent half-times (i.e, exponential series analysis). To avoid the limitations of this technique, we have formulated a method that combines analysis by moments of distribution with transfer function techniques, which makes the analysis independent of any mechanistic model. This method is an improvement over previous methods because it allows better comparison of gas exchange experiments and better prediction of gas exchange in the formulation of diver decompression schedules. Measurements of radioxenon uptake and elimination in two areas of a dog are presented. Analysis of the same organ response by both methods showed a fivefold variation in derived exponential time constants compared with less than 10% variation among moment descriptors.

[The effect of yttrium-90 radio synovectomy on the intra-articular xenon-133 clearance in rheumatic joint diseases]. / Der Einfluss der Yttrium-90-Radiosynoviorthese auf die intraarikuläre Xenon-133-Clearance bei rheumatischen Gelenkerkrankungen.

Nine inflamed knee joints out of eight patients were examined before and after intraarticular injection of yttrium-90 silicate colloid, in order to find out the effect of radiation synovectomy on the perfusion of the joints. These joints and the untreated contralateral knee joints were examined as to the clinical status and the fast component Tf of the xenon-133 clearance was determined as a means of the perfusion of the synovium. A linear correlation exists between the clinical status of the knee joint and the perfusion as determined by Tf measured three months before and after injection of yttrium-90. Also the joints without any radiation synovectomy showed this correlation. Three months after radiation synovectomy with yttrium-90 there was still no therapeutic effect apparent in the majority of the inflamed knee joints (seven out of nine). At this stage there still existed a hyperaemia. Therefore, it seems that on the whole three months are too short a period to judge the therapeutic effect.

[Xenon exhalation measurements in the diagnosis of bladder diseases]. / Die Xenon-Exhalationsmessung im Rahmen der Diagnostik von Harnblasenerkrankungen

Using an exhalameter radioactive marked Xenon, absorbed following bladder instillation, was determined in the expired air of 111 patients. In 65 of these patients the usual criteria in the diagnosis of bladder diseases were simultaneously ascertained. The functional disturbances of the epithelium of the bladder obtained through 133Xenon exhalation measurements did not correlate with the morphological changes. Especially in the so-called "Reizblase" (irritable bladder) functional disturbances were found without a morphological equivalent. Compared with the low Xenon exhalation found in patients with healthy urinary bladders there is a marked Xenon exhalation increase (10-15 times) in inflammation of the urinary bladder. In our opinion Xenon exhalation measurements is at the present time the most sensitive method for determining inflammatory diseases of the urinary bladder.