Biofilm-derived spores of Clostridioides (Clostridium) difficile exhibit increased thermotolerance compared to planktonic spores.

Biofilm-derived spores of strains of four ribotypes (001, 020, 027 & 078) of Clostridioides (Clostridium) difficile were found to exhibit increased thermotolerance compared to spores produced in planktonic culture. In addition, 'thick' and 'thin' exosporium morphotypes described previously were visualised by electron microscopy in both biofilm and planktonic spores.

Investigating the effect of supplementation on Clostridioides (Clostridium) difficile spore recovery in two solid agars.

BACKGROUND: A variety of supplemented solid media are used within Clostridium difficile research to optimally recover spores. Our study sought to investigate different media and additives, providing a method of optimised C. difficile spore recovery. Additionally, due to the results observed in the initial experiments, the inhibitory effects of three amino acids (glycine, l-histidine &l-phenylalanine) on C. difficile spore outgrowth were investigated. METHODS: Spores of five C. difficile strains (PCR ribotypes 001,015,020,027,078) were recovered on two commonly used solid media (BHI & CCEY, or cycloserine-cefoxitin egg yolk) supplemented with various concentrations of germinants (taurocholate, glycine & lysozyme). Agar-incorporation minimum inhibitory concentration (MIC) testing was carried out for glycine and taurocholate on vegetative cells and spores of all five strains. Additionally a BHI broth microassay method was utilised to test the growth of C. difficile in the presence of increasing concentrations (0,1,2,3,4%) of three amino acids (glycine,l-histidine,l-phenyalanine). RESULTS: CCEY agar alone and BHI supplemented with taurocholate (0.1/1%) provided optimal recovery for C. difficile spores. Glycine was inhibitory to spore recovery at higher concentrations, although these varied between the two media used. In agar-incorporated MIC testing, glycine concentrations higher than 2% (20 g/L) were inhibitory to both C. difficile spore and vegetative cell growth versus the control (mean absorbance = 0.33 ±â€¯0.02 vs 0.12 ±â€¯0.01) (P < 0.001). This indicates a potential mechanism whereby glycine interferes with vegetative cell growth. Further microbroth testing provided evidence of inhibition by two amino acids other than glycine, l-histidine and l-phenylalanine. CONCLUSIONS: We provide two media for optimal recovery of C. difficile spores (CCEY alone and BHI supplemented with 0.1/1% taurocholate). CCEY is preferred for isolation from faecal samples. For pure cultures, either CCEY or supplemented BHI agar are appropriate. The inhibitory nature of three amino acids (glycine,l-histidine,l-phenylalanine) to C. difficile vegetative cell proliferation is also highlighted.

Online self-exclusion from multiple gambling venues: Stakeholder co-design of a usable and acceptable self-directed website.

Gambling self-exclusion programs are under-utilised and barriers to entry include shame and embarrassment with face-to-face registration, and complex and effortful procedures. The current study aimed to facilitate self-exclusion from gambling venues via an online self-directed website. A co-design approach was used to elicit key stakeholders' perspectives on required website features, functionality, and to identify variables potentially impacting on development and implementation. Semi-structured focus groups and interviews were conducted across four stakeholders (N = 25): self-exclusion end users (consumers, n = 5), gambling counsellors (n = 7), venue staff (n = 6), and policy makers (n = 7). Overall, stakeholder perspectives were consistent with content analysis indicating the importance of website user-friendliness, flexibility, supportiveness, and trustworthiness. Importantly, these attributes were linked to target end users': perceived vulnerabilities, diverse backgrounds and individual expectations. Participants believed that the entire self-exclusion process should be conducted online, including identity verification, whilst expecting high-level data security measures to protect their personal privacy. A separate webpage was also suggested containing relevant information and links to additional help services, such as counselling. This study describes an adaptable co-design framework for developing a usable and acceptable self-exclusion website. Future studies should empirically test system usability and acceptability to refine and maximise system uptake upon implementation. Findings may have broader implications for digital health intervention design.

Investigating the transient and persistent effects of heat on Clostridium difficile spores.

Purpose. Clostridium difficile spores are extremely resilient to high temperatures. Sublethal temperatures are associated with the 'reactivation' of dormant spores, and are utilized to maximize C. difficile spore recovery. Spore eradication is of vital importance to the food industry. The current study seeks to elucidate the transient and persisting effects of heating C. difficile spores at various temperatures.Methods. Spores of five C. difficile strains of different ribotypes (001, 015, 020, 027 and 078) were heated at 50, 60 and 70-80 °C for 60 min in phosphate-buffered saline (PBS) and enumerated at 0, 15, 30, 45 and 60 min. GInaFiT was used to model the kinetics of spore inactivation. In subsequent experiments, spores were transferred to enriched brain heart infusion (BHI) broths after 10 min of 80 °C heat treatment in PBS; samples were enumerated at 90 min and 24 h.Results. The spores of all strains demonstrated log-linear inactivation with tailing when heated for 60 min at 80 °C [(x̄=7.54±0.04 log10 vs 4.72±0.09 log10 colony-forming units (c.f.u.) ml- 1; P<0.001]. At 70 °C, all strains except 078 exhibited substantial decline in recovery over 60 min. Interestingly, 50 °C heat treatment had an inhibitory effect on 078 spore recovery at 0 vs 60 min (7.61±0.06 log10 c.f.u. ml- 1 vs 6.13±0.05 log10 c.f.u. ml- 1; P<0.001). Heating at 70/80 °C inhibited the initial germination and outgrowth of both newly produced and aged spores in enriched broths. This inhibition appeared to be transient; after 24 h vegetative counts were higher in heat-treated vs non-heat-treated spores (x̄=7.65±0.04 log10 c.f.u. ml- 1 vs 6.79±0.06 log10 c.f.u. ml- 1; P<0.001).Conclusions. The 078 spores were more resistant to the inhibitory effects of higher temperatures. Heat initially inhibits spore germination, but the subsequent outgrowth of vegetative populations accelerates after the initial inhibitory period.

Distinctive patterns of BCL6 molecular alterations and their functional consequences in different subgroups of diffuse large B-cell lymphoma.

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) has revealed biologically and prognostically distinct subgroups: germinal center B-cell-like (GCB), activated B-cell-like (ABC) and primary mediastinal (PM) DLBCL. The BCL6 gene is often translocated and/or mutated in DLBCL. Therefore, we examined the BCL6 molecular alterations in these DLBCL subgroups, and their impact on BCL6 expression and BCL6 target gene repression. BCL6 translocations at the major breakpoint region (MBR) were detected in 25 (18.8%) of 133 DLBCL cases, with a higher frequency in the PM (33%) and ABC (24%) subgroups than in the GCB (10%) subgroup. Translocations at the alternative breakpoint region (ABR) were detected in five (6.4%) of 78 DLBCL cases, with three cases in ABC and one case each in the GCB and the unclassifiable subgroups. The translocated cases involved IgH and non-IgH partners in about equal frequency and were not associated with different levels of BCL6 mRNA and protein expression. BCL6 mutations were detected in 61% of DLBCL cases, with a significantly higher frequency in the GCB and PM subgroups (>70%) than in the ABC subgroup (44%). Exon-1 mutations were mostly observed in the GCB subgroup. The repression of known BCL6 target genes correlated with the level of BCL6 mRNA and protein expression in GCB and ABC subgroups but not with BCL6 translocation and intronic mutations. No clear inverse correlation between BCL6 expression and p53 expression was observed. Patients with higher BCL6 mRNA or protein expression had a significantly better overall survival. The biological role of BCL6 in translocated cases where repression of known target genes is not demonstrated is intriguing and warrants further investigation.

Microbiologic factors affecting Clostridium difficile recurrence.

BACKGROUND: Recurrent Clostridium difficile infection (rCDI) places a huge economic and practical burden on healthcare facilities. Furthermore, rCDI may affect quality of life, leaving patients in an rCDI cycle and dependant on antibiotic therapy. AIMS: To discuss the importance of microbiologic factors in the development of rCDI. SOURCES: Literature was drawn from a search of PubMed from 2000 onwards with the search term 'recurrent Clostridium difficile infection' and further references cited within these articles. CONTENT: Meta-analyses and systematic reviews have shown that CDI and rCDI risk factors are similar. Development of rCDI is attendant on many factors, including immune status or function, comorbidities and concomitant treatments. Studies suggest that poor bacterial diversity is correlated with clinical rCDI. Narrow-spectrum gut microflora-sparing antimicrobials (e.g. surotomycin, cadazolid, ridinilazole) are in development for CDI treatment, while microbiota therapeutics (faecal microbiota transplantation, nontoxigenic C. difficile, stool substitutes) are increasingly being explored. rCDI can only occur when viable C. difficile spores are present, either within the gut lumen after infection or when reacquired from the environment. C. difficile spore germination can be influenced by gut environmental factors resulting from dysbiosis, and spore outgrowth may be affected stage by some antimicrobials (e.g. fidaxomicin, ramoplanin, oritavancin). IMPLICATIONS: rCDI is a significant challenge for healthcare professionals, requiring a multifaceted approach; optimized infection control to minimize reinfection; C. difficile-targeted antibiotics to minimize dysbiosis; and gut microflora restoration to promote colonization resistance. These elements should be informed by our understanding of the microbiologic factors involved in both C. difficile itself and the gut microbiome.

Modelling affective pain in mice: Effects of inflammatory hypersensitivity on place escape/avoidance behaviour, anxiety and hedonic state.

BACKGROUND: The place escape/avoidance paradigm (PEAP) has been used to assess the affective component of pain in rats. Using the Complete Freund's Adjuvant (CFA) model of inflammatory pain, the current study aimed at developing a mouse version of PEAP and investigating the relation between PEAP and other behavioural responses, namely anxiety-like behaviour, locomotor activity, and hedonic state. NEW METHOD: A novel paradigm assessing the affective component of pain in mice was developed by modifying the setup known from rat studies: Animals were forced to stay 2 × 5 min in the light and the dark area of a box while being stimulated with a suprathreshold filament on the untreated or treated paw, respectively. This was followed by a 30-min test with unrestricted movement. Anxiety-like behaviour, locomotor activity, and hedonic state were assessed with the elevated zero maze (EZM), an open field setup, and a saccharin preference test, respectively, and correlated with the PEAP behaviour to examine potentially confounding parameters of the novel paradigm. RESULTS: In the PEAP, CFA-treated animals spent more time in the light area. CFA also increased anxiety-like behaviour significantly, whereas locomotor activity was unaffected. A significant, albeit modest, reduction in saccharin preference was observed. PEAP responses showed no significant correlations with any other behavioural measure. COMPARISON WITH EXISTING METHOD AND CONCLUSIONS: The PEAP results suggest that this paradigm might be successfully applied in mice to study affective pain. CFA treatment was associated with increased anxiety-like behaviour and anhedonia; however, this appeared unrelated to the PEAP responses.

Identification of amino acid residues in GluR1 responsible for ligand binding and desensitization.

Although GluR1(o) and GluR3(o) are homologous at the amino acid level, GluR3(o) desensitizes approximately threefold faster than GluR1(o). By creating chimeras of GluR1(o) and GluR3(o) and point amino acid exchanges in their S2 regions, two residues were identified to be critical for GluR1(o) desensitization: Y716 and the R/G RNA-edited site, R757. With creation of the double-point mutant (Y716F, R757G)GluR1(o), complete exchange of the desensitization rate of GluR1(o) to that of GluR3(o) was obtained. In addition, both the potency and affinity of the subtype-selective agonist bromohomoibotenic acid were exchanged by the Y716F mutation. A model is proposed of the AMPA receptor binding site whereby a hydrogen-bonding matrix of water molecules plays an important role in determining both ligand affinity and receptor desensitization properties. Residues Y716 in GluR1 and F728 in GluR3 differentially interact with this matrix to affect the binding affinity of some ligands, providing the possibility of developing subtype-selective compounds.

Expression of nanomolar-affinity binding sites for melatonin in Syrian hamster RPMI 1846 melanoma cells.

Pharmacological studies indicate that Syrian hamster melanoma (RPMI 1846) cells possess a melatonin binding site similar to that found in normal hamster cells. A high correlation was observed for a series of compounds between the Ki in hamster hypothalamic membranes vs. RPMI 1846 membranes (r = 0.94, slope = 0.93, P less than 0.01, n = 14). Scatchard analysis of saturation binding of 2-[125I]-iodomelatonin to membranes (at 0 degrees C) indicated: Kd = 0.89 +/- 0.08 nM, Bmax = 6.2 +/- 2.9 fmol/mg protein (n = 3). Melatonin did not alter basal or forskolin-stimulated adenylate cyclase activity in RPMI 1846 membranes or intact cells. Therefore, in contrast to the picomolar-affinity receptor for melatonin in the mammalian hypothalamus and pars tuberalis, the putative nanomolar-affinity receptor is not coupled to adenylate cyclase. The RPMI 1846 cell line provides a useful model system for further studies of signal transduction via the nanomolar-affinity site for melatonin.

Audit of local recurrence rates following 'ultra'-conservative surgery for invasive breast cancer--a boost to the breast?

BACKGROUND: Conservative breast surgery with postoperative radiotherapy and appropriate systemic therapy is associated with similar outcomes when compared with mastectomy. The reported 5 year local recurrence rate varies between 3% and 15%. We prefer a more conservative 'complete' local excision rather than 'wide' local excision combined with post-operative radical radiotherapy and tumour bed boost with the aim of achieving optimal cosmesis. AIMS: Our review was undertaken to assess whether or not this 'ultra' conservative approach was compromising long-term local control. METHODS: Case notes and pathology reports of patients who underwent conservative surgery for breast cancer from January 1983 to February 2001 were accessed for this audit. Patient demographic data and tumour characteristics were noted. The primary outcome data were the number of local recurrences following invasive breast cancer at 5 and 10 years and the distance from the tumour to the closest margin of excision. RESULTS: At 5 and 10 years there were 16/451 and 5/124 local recurrences, with a local recurrence rate of 3.5% (95% CI, 1.7-4.7%) and 4.1% (95% CI, 0.47-6.5%), respectively. Complete data with regards to the closest histological margin of excision were available in 423 patients. One hundred and sixty-five patients (39%) had their tumours excised with a distance of less than 1 mm to the closest margin. Nearly, all tumours (97.8%) were excised with the distance to the closest margin less than 1 cm and 81% with 5 mm or less. CONCLUSION: It is possible to achieve low local recurrence rates after very conservative surgery for breast cancer when this is combined with radical radiotherapy and an additional tumour bed boost.

Rearrangements of chromosome band 1p36 in non-Hodgkin's lymphoma.

We studied 850 consecutive cases of histologically ascertained pretreatment non-Hodgkin's lymphoma with cytogenetically abnormal clones. The diagnostic karyotypes revealed that 12% of these cases exhibited structural rearrangements involving chromosome band 1p36. Here, we describe the karyotypes of 53 cases containing a 1p36 rearrangement [often involving translocations of unknown material and presented as add(1)(p36)]. We used fluorescence in situ hybridization to determine the origin of the translocation partners. We report three different recurrent translocations involving 1p36. These include der(1)t(1;1)(p36;q21) (three cases), der(1)t(1;1)(p36;q25) (three cases), and der(1)t(1;9)(p36;q13) (four cases). Using cytogenetic and fluorescence in situ hybridization analyses, we have resolved the translocation partners in 31 cases. Rearrangements of band 1p36 were found among different histopathological subtypes. Alterations of 1p36 never occurred as a sole abnormality, and in 42 of 53 cases, alterations of the band 14q32 were observed. The t(14;18)(q32;q21) translocation was present in 35 cases. The significantly high occurrence of 1p36 breakpoint in structural rearrangements and its involvement in recurrent translocations suggest that the region is bearing gene(s) that are important in lymphomagenesis. Our study also showed that cytogenetically evident deletions were frequent in chromosome 1p, almost always involving the p36 region, whereas duplications were rare and never encompassed the p36 region. Chromosome band 1p36 harbors many candidate tumor suppressor genes, and we propose that one or more of these genes might be deleted or functionally disrupted as a molecular consequence of the rearrangements, thus contributing to lymphomagenesis.

Fast neutron therapy for soft tissue sarcoma.

Seventy-nine patients with soft tissue sarcoma were treated with fast neutron therapy at the Hammersmith Hospital, MRC Cyclotron Unit. Sixty-six of these, treated between 1971 and 1983 were assessable. The histology was reviewed and graded in 82% of cases and tumors divided into groups according to maximum diameter. In sixteen patients, who were irradiated following complete macroscopic removal of tumor, there was 94% local control and 86% survived 5 years. Of the 50 patients who had gross tumors present 62% were greater than 10 cm in diameter, and 20 were recurrent after previous radiotherapy or surgery or both. Sixty-eight per cent of gross tumors completely regressed and local control was 52%. The main cause of death was metastatic spread, and median survival was 63 months for Grade 1 patients, 9 months for Grade 2, and 7 months for Grade 3. Thus, there was a significant advantage to patients with Grade 1 tumor but little difference between Grades 2 and 3. Twenty-seven patients experienced late complications of treatment, 67% of which involved the skin predominantly and were related to the low energy of neutrons used. Seventeen of the 27 had received previous radiotherapy. Neutron therapy given in this dose and fractionation produced a higher local control rate than photon therapy, but complications were more frequent. Since these mainly involved the skin a lower level of complications may be anticipated using higher energy neutrons which will have a more even distribution of dose and lower skin dosage. Forty-eight per cent of patients developed metastatic disease, indicating the need for effective systemic therapy, especially in Grades 2 and 3 tumors.

Translocation t(1;3)(p36.3;q25) is a nonrandom aberration in epithelioid hemangioendothelioma.

The cytogenetic findings for two epithelioid hemangioendotheliomas are reported. An identical chromosomal translocation involving chromosomes 1 and 3 [t(1;3)(p36.3;q25)] was detected in both cases of epithelioid hemangioendothelioma, possibly representing a characteristic rearrangement for this histopathologic entity. The presence of clonal karyotypic abnormalities supports a neoplastic origin for the epithelioid variant of hemangioendothelioma. Identification of the 1;3 translocation may be useful diagnostically. Should additional studies confirm these data, this could lead to the identification of the gene(s) central to this neoplastic process.

Characterization of the 1H-cyclopentapyrimidine-2,4(1H,3H)-dione derivative (S)-CPW399 as a novel, potent, and subtype-selective AMPA receptor full agonist with partial desensitization properties.

(S)-CPW399 (2b) is a novel, potent, and subtype-selective AMPA receptor full agonist that, unlike (S)-willardiine and related compounds, in mouse cerebellar granule cells, stimulated an increase in [Ca(2+)](i), and induced neuronal cell death in a time- and concentration-dependent manner. Compound 2b appears to be a weakly desensitizing, full agonist at AMPA receptors and therefore represents a new pharmacological tool to investigate the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitization.

Characterization of two alternatively spliced forms of a metabotropic glutamate receptor in the central nervous system of the rat.

Amplification of complementary DNA by the polymerase chain reaction and anti-peptide antibodies were used to characterize the expression of two alternatively spliced forms of a metabotropic glutamate receptor (mGluR1 alpha and mGluR1 beta) in the central nervous system of the rat. Polymerase chain reaction analysis showed that mGluR1 alpha was the predominate of the two forms in the cerebellum, diencephalon, mesencephalon, olfactory bulb and brainstem, while mGluR1 beta was the major form present in the hippocampus. Approximately equal amounts of the two receptors were expressed in the cerebral cortex, septum and striatum. Immunochemical analyses of the two receptors were conducted in the rat cerebellum and hippocampus. An mGluR1 alpha-specific antibody labelled a protein with a relative molecular weight of 146,000 on immunoblots of the hippocampus and cerebellum. Immunoblot analysis of the developmental expression of mGluR1 alpha in the hippocampus and cerebellum demonstrated that in both structures, the levels of mGluR1 alpha were at or near their maximum levels in the adult brain. In contrast, two mGluR1 beta-specific antibodies failed to detect mGluR1 beta on immunoblots of brain tissue, thus precluding an immunocytochemical analysis of this receptor. Although low levels of a higher-molecular weight protein, possibly a dimeric form of mGluR1 beta were seen with one of the mGluR1 beta-specific antibodies, we hypothesize that some of the mGluR1 beta present in brain tissue may undergo proteolytic cleavage of the carboxy terminus. Immunocytochemical analysis of mGluR1 alpha showed that very high levels of this receptor were expressed in Purkinje cell bodies and dendrites. In the granule cell layer, some Golgi neurons were immunostained. The granule cells were not labelled. In the hippocampus, mGluR1 alpha immunoreactivity was present in interneurons of the stratum oriens and the dentate hilar region. Double-labelling studies demonstrated that these interneurons were also immunopositive for the neuropeptide somatostatin. The presence of mGluR1 alpha in cells of the hippocampus that are associated with the release of somatostatin, suggest that this receptor could play a role in regulating hippocampal excitability in both normal and epileptic tissues.

Involvement of multiple sulfhydryl groups in melatonin signal transduction in chick brain.

To gain insight into the molecular mechanism underlying melatonin binding and signal transduction in the chick brain, we have investigated the role of -SH groups, using a sulfhydryl alkylating reagent N-ethylmaleimide (NEM). At least two -SH groups are involved in the formation of the receptor-G protein complex: one is sensitive to and the other relatively insensitive to NEM. Alkylation of the sensitive group selectively abolishes high affinity binding of 2-[125I]iodomelatonin ([125I]MEL), similar to the effect induced by GTP, thus leading to a complete loss of sensitivity to nucleotides. Modification of both groups causes a marked reduction in binding capacity. Agonists with high affinity, but not other compounds with low affinity for the melatonin receptor, protect against alkylation by NEM. GTP gamma s does not significantly alter the reactivity of -SH groups towards NEM, but agonist-protected receptors remain sensitive to this nucleotide. Moreover, NEM pretreatment blocks the inhibitory effect of melatonin on forskolin-stimulated adenylate cyclase activity in chick brain. These data suggest that the -SH group modulating agonist affinity may lie within the coupling domain between the receptor and G protein but outside of the GTP binding site. In addition, sulfhydryl groups are essential for melatonin binding and signal transduction in chick brain.

Familial inverted duplication 7p.

A 10-month-old female with developmental delay and failure to thrive was referred for genetic evaluation as part of an adoption assessment. Physical exam showed a mildly beaked nose and clinodactyly, but otherwise nothing remarkable. Chromosome analysis showed an inverted duplication of the p12.2-->p13 portion of chromosome 7[(46,XX, dup(7)(p13p12.2)]. The proposita's older brother, mother, and grandmother were cognitively delayed and had the same chromosome 7 duplication. A review of the literature showed no other cases involving this exact duplication.

Transcriptional regulation of MMP-9 expression in stromal cells of human giant cell tumor of bone by tumor necrosis factor-alpha.

We determined whether certain factor(s) secreted by multinucleated giant cells, which is of monocyte/macrophage lineage in giant cell tumor of bone (GCT), regulate the induction of matrix metalloproteinase (MMP)-9 expression in mononucleated stromal cells. Our data derived using enzyme linked immunosorbant assays (ELISAs) suggest that the GCT cells in primary culture produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha). Further, the MMP-9 expression in GCT primary cultures was partially abrogated by neutralizing antibody to TNF-alpha, suggesting that TNF-alpha secretion by the multinucleated giant cells may be one of the factors responsible for the production of MMP-9 by the stromal cells in vivo. In order to confirm this we examined the role of TNF-alpha on the induction of MMP-9 expression in bone GCT stromal cells. These cells express MMP-2, but not MMP-9. However, treatment of these cells with TNF-alpha induced the expression of MMP-9 in a concentration-dependent manner. Kinetic experiments revealed that the secretion of MMP-9 peaked 12 h post TNF-alpha stimulation. Immunofluorescence studies confirmed the expression of MMP-9 after stimulation of GCT stromal cells with TNF-alpha. Further, TNF-alpha-induced MMP-9 expression was completely blocked with neutralizing antibody to TNF-alpha, thereby demonstrating the specificity. In addition, the induction of MMP-9 expression by TNF-alpha was completely abrogated in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that de novo protein synthesis may be required. Nuclear run-on analysis demonstrated that treatment of GCT stromal cells significantly enhanced the MMP-9 gene transcription. Together, our data suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. These studies also suggest the existence of an essential cell-cell interaction in the regulation of MMP-9 expression in GCT.

AMPA receptor mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization.

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) excitotoxicity was examined in cultured neocortical neurons using the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to measure cell viability. Neurons were exposed to AMPA at different culture periods during development of the neurons. In order to describe the pharmacology of AMPA-mediated toxicity, several glutamate receptor antagonists were used: MK-801, NS 394, NBQX, GYKI 52466, GYKI 53405 and GYKI 53655. Increased excitotoxicity was observed when cortical neurons cultured for 5, 8 and 12 days in vitro (DIV) were exposed to a high concentration of AMPA (500 microM) for 6 h. However, only at DIV 12 was part of the toxicity mediated directly through AMPA receptors since 10 microM MK-801 blocked all AMPA toxicity at DIV 5 and 8, but only some of the AMPA response at DIV 12. This indicated that NMDA receptors were being activated, causing some of the observed toxicity. The high dose of AMPA was not sufficient to damage all neurons since 59% remained viable after exposure to AMPA even for neurons that were cultured for 12 DIV. Since it is known that both glutamate and AMPA activate AMPA receptors with a fast and rapidly desensitizing response, this could explain the relatively low toxicity produced by 500 microM AMPA. This was investigated by blocking AMPA receptor desensitization with cyclothiazide. Using a lower concentration (25 microM) of AMPA, addition of 50 microM cyclothiazide increased the AMPA induced excitotoxicity in cultured cortical neurons at all DIV except for DIV 2. This combination of AMPA + cyclothiazide yielded 77% cell death for DIV 12 cultures. In contrast to the results observed with 500 microM AMPA, the neurotoxicity mediated directly by AMPA receptors when desensitization was blocked was seen as early as 5 DIV since 10 microM MK-801 did not completely block the response whereas 10 microM NBQX did. The 2,3-benzodiazepine GYKI compounds, which have been reported to be selective non-competitive AMPA receptor antagonists, were here observed to block the AMPA toxicity with the following rank order: GYKI 53655 > GYKI 52466 > or = GYKI 53405, which is in agreement with their published potencies.

Molecular determinants of desensitization and assembly of the chimeric GABA(A) receptor subunits (alpha1/gamma2) and (gamma2/alpha1) in combinations with beta2 and gamma2.

Two gamma-aminobutyric acid(A) (GABA(A)) receptor chimeras were designed in order to elucidate the structural requirements for GABA(A) receptor desensitization and assembly. The (alpha1/gamma2) and (gamma2/alpha1) chimeric subunits representing the extracellular N-terminal domain of alpha1 or gamma2 and the remainder of the gamma2 or alpha1 subunits, respectively, were expressed with beta2 and beta2gamma2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (alpha1/gamma2)beta2 and (alpha1/gamma2)beta2gamma2 but not the (gamma2/alpha1)beta2 and (gamma2/alpha1)beta2gamma2 subunit combinations formed functional receptor complexes as shown by whole-cell patch-clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (alpha1/gamma2)-containing receptors was pronounced, as opposed to the staining of the (gamma2/alpha1)-containing receptors, which was only slightly higher than background. To explain this, the (alpha1/gamma2) and (gamma2/alpha1) chimeras may act like alpha1 and gamma2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (alpha1/gamma2) chimeric subunit had characteristics different from the alpha1 subunit, since the (alpha1/gamma2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch-clamp recordings, which was independent of whether the chimera was expressed in combination with beta2 or beta2gamma2. Surprisingly, the (alpha1/gamma2)(gamma2/alpha1)beta2 subunit combination did desensitize, indicating that the C-terminal segment of the alpha1 subunit may be important for desensitization. Moreover, desensitization was observed for the (alpha1/gamma2)beta2gamma2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.