Etomidate Impairs Long-Term Potentiation In Vitro by Targeting α5-Subunit Containing GABAA Receptors on Nonpyramidal Cells.

Previous experiments using genetic and pharmacological manipulations have provided strong evidence that etomidate impairs synaptic plasticity and memory by modulating α5-subunit containing GABAA receptors (α5-GABAARs). Because α5-GABAARs mediate tonic inhibition (TI) in hippocampal CA1 pyramidal cells and etomidate enhances TI, etomidate enhancement of TI in pyramidal cells has been proposed as the underlying mechanism (Martin et al., 2009). Here we tested this hypothesis by selectively removing α5-GABAARs from pyramidal neurons (CA1-pyr-α5-KO) and comparing the ability of etomidate to enhance TI and block LTP in fl-α5 (WT), global-α5-KO (gl-α5-KO), and CA1-pyr-α5-KO mice. Etomidate suppressed LTP in slices from WT and CA1-pyr-α5-KO but not gl-α5-KO mice. There was a trend toward reduced TI in both gl-α5-KO and CA1-pyr-α5-KO mice, but etomidate enhanced TI to similar levels in all genotypes. The dissociation between effects of etomidate on TI and LTP in gl-α5-KO mice indicates that increased TI in pyramidal neurons is not the mechanism by which etomidate impairs LTP and memory. Rather, the ability of etomidate to block LTP in WT and CA1-pyr-α5-KO mice, but not in gl-α5-KO mice, points toward α5-GABAARs on nonpyramidal cells as the essential effectors controlling plasticity in this in vitro model of learning and memory.

Crystallization and quaternary structure analysis of the NAD(+)-dependent leucine dehydrogenase from Bacillus sphaericus.

The NAD(+)-dependent leucine dehydrogenase from Bacillus sphaericus has been crystallized by the hanging drop method of vapour diffusion, using ammonium sulphate as the precipitant. The crystals belong to the tetragonal system and are in space group I4, with unit cell dimensions of a = b = 138.4 A and c = 121.8 A. Considerations of the values of Vm, the space group symmetry and an analysis of a self-rotation function calculated on a preliminary data set collected to 3 A resolution show that the asymmetric unit contains a dimer with the twofold axis perpendicular to the crystallographic four fold, indicating that the quaternary structure of this enzyme is octameric. Leucine dehydrogenase belongs to a superfamily of amino acid dehydrogenases which display considerable differences in amino acid specificity and elucidation of its three-dimensional structure should enable the molecular basis of this differential specificity to be examined in detail.

An international outbreak of salmonellosis associated with raw almonds contaminated with a rare phage type of Salmonella enteritidis.

During the winter of 2000 to 2001, an outbreak due to Salmonella Enteritidis (SE) phage type 30 (PT30), a rare strain, was detected in Canada. The ensuing investigation involved Canadian and American public health and food regulatory agencies and an academic research laboratory. Enhanced laboratory surveillance, including phage typing and pulsed-field gel electrophoresis, was used to identify cases. Case questionnaires were administered to collect information about food and environmental exposures. A case-control study with 16 matched case-control pairs was conducted to test the hypothesis of an association between raw whole almond consumption and infection. Almond samples were collected from case homes, retail outlets, and the implicated processor, and environmental samples were collected from processing equipment and associated farms for microbiological testing. One hundred sixty-eight laboratory-confirmed cases of SE PT30 infection (157 in Canada, 11 in the United States) were identified between October 2000 and July 2001. The case-control study identified raw whole almonds as the source of infection (odds ration, 21.1; 95% confidence interval, 3.6 to infinity). SE PT30 was detected in raw whole natural almonds collected from home, retail, distribution, and warehouse sources and from environmental swabs of processing equipment and associated farmers' orchards. The frequent and prolonged recovery of this specific organism from a large agricultural area was an unexpected finding and may indicate significant diffuse contamination on these farms. Identification of almonds as the source of a foodborne outbreak is a previously undocumented finding, leading to a North American recall of this product and a review of current industry practices.

Etomidate blocks LTP and impairs learning but does not enhance tonic inhibition in mice carrying the N265M point mutation in the beta3 subunit of the GABA(A) receptor.

Enhancement of tonic inhibition mediated by extrasynaptic α5-subunit containing GABAA receptors (GABAARs) has been proposed as the mechanism by which a variety of anesthetics, including the general anesthetic etomidate, impair learning and memory. Since α5 subunits preferentially partner with ß3 subunits, we tested the hypothesis that etomidate acts through ß3-subunit containing GABAARs to enhance tonic inhibition, block LTP, and impair memory. We measured the effects of etomidate in wild type mice and in mice carrying a point mutation in the GABAAR ß3-subunit (ß3-N265M) that renders these receptors insensitive to etomidate. Etomidate enhanced tonic inhibition in CA1 pyramidal cells of the hippocampus in wild type but not in mutant mice, demonstrating that tonic inhibition is mediated by ß3-subunit containing GABAARs. However, despite its inability to enhance tonic inhibition, etomidate did block LTP in brain slices from mutant mice as well as in those from wild type mice. Etomidate also impaired fear conditioning to context, with no differences between genotypes. In studies of recombinant receptors expressed in HEK293 cells, α5ß1γ2L GABAARs were insensitive to amnestic concentrations of etomidate (1 µM and below), whereas α5ß2γ2L and α5ß3γ2L GABAARs were enhanced. We conclude that etomidate enhances tonic inhibition in pyramidal cells through its action on α5ß3-containing GABAA receptors, but blocks LTP and impairs learning by other means - most likely by modulating α5ß2-containing GABAA receptors. The critical anesthetic targets underlying amnesia might include other forms of inhibition imposed on pyramidal neurons (e.g. slow phasic inhibition), or inhibitory processes on non-pyramidal cells (e.g. interneurons).

Guidelines and care pathways for genetic diseases: the Scottish collaborative project on tuberous sclerosis.

In Scotland a national audit project has been undertaken to devise evidence-based guidelines for the clinical management of patients with tuberous sclerosis (TS), a dominantly inherited multisystem disorder. In order to facilitate the audit and use of these guidelines a 'Care Pathway' was devised to form the patient records. We describe the process of guideline development for TS and our TS Care Pathway.

A method for measuring cyto-adherence on frozen tissue sections.

A simple method for quantifying the cells adhering to frozen sections of mouse spleen using a microscope equipped with a drawing tube attachment, and a planimeter is described. It is a straightforward and reproducible technique which was been preliminarily employed to study optimal binding conditions for Esheep and EoxAC.

Proposed pathogenic mechanism for the diarrhea associated with human intestinal spirochetes.

Spirochetes resembling Brachyspira aalborgi were found in the feces and rectal biopsies of a patient with persistent diarrhea. Although the organism failed to grow on bacteriologic media, it was found attached to the surfaces of the epithelial cells on the rectal lumen. Blunting and destruction of the cellular microvilli was evident. These induced pathologic cell surface changes, together with the presence of intracellular bacteria in the cells of the rectal colon, suggest a pathogenic mechanism for the persistent diarrhea often associated with this condition. Both the spirochetosis and clinical symptoms disappeared on treatment with metronidazole.

Ultrastructure of Legionella pneumophila.

Eleven lung samples positive for Legionnaires' disease, 12 strains of Legionella pneumophila cultured on various bacteriological media, and one strain growth in the yolk sac of fertile hens' eggs were examined by negative staining, thin sectioning, and scanning electron microscopy. All organisms studied were ultrastructurally similar irrespective of strain, source, or method of cultivation, presenting mainly as short rods, 0.6 x 1.5 micrometer, with tapered ends, though long forms and filaments were also evident. In this they resembled typical Gram-negative organisms. Division was by non-septate binary fission, and the cell wall was composed of two triple-unit membranes with morphological evidence of a peptidoglycan layer. The bacterial cytoplasm was rich in ribosomes and nuclear elements and often contained vacuoles. No acid polysaccharides or bacterial appendages were detected surrounding the organisms. In lung tissue and yolk sac membranes, the organisms replicated within the cytoplasm of infected cells and in the intercellular spaces and were specifically identified in thin sections by immunoferritin techniques.

Long-term storage of blood in liquid nitrogen, and the response of the recovered red cells to haemagglutination by viruses.

Human and chicken blood were snap-frozen as droplets in liquid ntirogen. Storage in the vapour phase liquid nitrogen container for nine months followed by rapid thawing resulted in approximately 10% haemolysis of the red cells. The ability of the thawed red cells to haemagglutinate viruses was unimpaired by the manipulations.

Detection of virus particles by electron microscopy with polyacrylamide hydrogel.

The use of lyphogel to concentrate the number of virus particles in specimens for electron microscopic examination was studied in parallel with ultracentrifugation. One hundred faecal and urine samples were compared. Both methods had a similar sensitivity. Lyphogel was economical, simple, and rapid in use; in contrast to ultracentrifugation, it required relatively little material. The procedure could be done within a safety cabinet, and virus particles were morphologically undamaged by the process.

Electron microscopic evidence of flagella and pili on Legionella pneumophila.

Twenty-one strains of Legionella pneumophila, representing the six known serotypes of the organism, cultured on various bacteriological media and in the yolk sacs of fertile hens' eggs were examined by negative stain electron microscopy for flagella and pili. These appendages were usually observed after cultivation on media capable of inducing an early profuse growth of the organisms.

Identification of Legionella pneumophila antigens and antibodies by immunoferritin electron microscopy.

Material from 18 lungs positive for Legionella pneumophila and 21 strains of legionella grown on bacteriological media or in the yolk sac of fertile hen's eggs were examined by direct negative stain and immunoferritin electronmicroscopy. The 18 lung samples and all preparations of cultivated organisms showed the presence of bacteria with the typical morphology of L. pneumophila. By immunoferritin electronmicroscopy, with specific antisera, it was possible to serogroup the organisms. The immunoferritin method in reverse made it possible to detect and titrate antibody in sera from patients. Direct and immunoferritin electronmicroscopy were as sensitive as immunofluorescent antibody tests for detecting the antigens and antibodies of Legionnaires' disease. Additional advantages of the ultrastructural technique are discussed.

Morphological response and growth characteristics of Legionella pneumophila exposed to ampicillin and erythromycin.

The morphological response of two strains of Legionella pneumophila to ampicillin 10 micrograms/ml and erythromycin 10 micrograms/ml in vitro was studied by electronmicroscopy, MIC estimations and viable counts. In the presence of ampicillin, discrete lesions appeared in the bacterial cell walls through which cytoplasmic contents extruded and lysis occurred. A few spheroplasts, together with minicells of 0.15-micron diameter, and apparently normal cells were present after exposure to ampicillin for several hours. Conversely, erythromycin initially resulted in inhibition of division and the formation of filamentous organisms. The cell walls of these filaments were eventually disrupted with numerous small membranous vesicles appearing on their surfaces. On further erythromycin treatment, breakage of the cell wall at a restricted number of sites occurred, leading to cell lysis. In the presence of erythromycin, a few morphologically normal cells were present but no spheroplasts or minicells were observed. Viable counts demonstrated that ampicillin killed the bacteria faster than erythromycin. Regrowth did not occur in the continued presence of either antibiotic, but after their removal regrowth was observed.

Opsonin-independent adherence and phagocytosis of Listeria monocytogenes by murine peritoneal macrophages.

Listeria monocytogenes adhered to and multiplied intracellularly in murine peritoneal macrophages in the absence of opsonins. The infective process in these cells was evaluated by viable bacterial cell colony counts of intracellular organisms and documented by transmission and scanning electron microscopy. Adherence of listeriae to macrophages involved surface interactions of the prokaryotic cell surface and eukaryotic cell membranes. Subsequent phagocytosis was seen to occur through a process in which host cell-derived pseudopodia surrounded and engulfed organisms leaving them within phagosomes in the cytoplasm of infected cells. This process of uptake of L. monocytogenes by macrophages occurred at 4 degrees C. Following invasion of the cell, escape of L. monocytogenes from the phagosome into the cytoplasm was initiated as early as 10 min into the infective process. Intracellular multiplication of bacteria continued for 8 h after inoculation at which point loss of adherent macrophages due to cell lysis was evident. The mean generation time of the organism in these cells was 58 min. The cellular and ultrastructural events of L. monocytogenes adherence to and phagocytosis by murine macrophages in the absence of antibody or complement have been defined.

The effect of antibiotics that inhibit cell-wall, protein, and DNA synthesis on the growth and morphology of Legionella pneumophila.

The response of Legionella pneumophila to antibiotics that inhibit cell-wall, protein and DNA synthesis was examined by electronmicroscopy, MIC estimations and viable counts. Ampicillin, cefotaxime, methicillin, erythromycin, rifampicin and ciprofloxacin, each used separately at 20 times their respective MIC values, showed activity against L. pneumophila in these studies. The inhibitors of cell-wall synthesis--ampicillin, cefotaxime and methicillin--effected the greatest bactericidal activity and induced the most extensive morphological changes, which included the formation of membranous lesions through which cytoplasmic contents were lost. In terms of ultrastructural damage and loss of viability, the inhibitors of protein and DNA synthesis were less effective than the antibiotics that acted on the microbial cell wall. Erythromycin- and rifampicin-treated cells possessed irregular membranes and were partially or fully lysed, whereas ciprofloxacin induced abnormally elongated organisms with intermittently lysed and detached inner membranes. These results illustrated the ability of antibiotics of putative clinical value, with diverse modes of action, to affect the ultrastructural cytology as well as the viability of L. pneumophila in vitro.

Morphological response of human rotavirus to ultra-violet radiation, heat and disinfectants.

The morphological damage induced in human rotavirus particles by exposure to ultraviolet (UV) radiation at a wavelength of 254 nm increased progressively with length of treatment. Exposure of the virus in suspension to 9000 ergs/cm2/s was sufficient to remove the smooth capsid layer from 50% of particles after 1 min and from all the virions within 10 min. By this time, the number of stain-penetrated or empty particles increased markedly, along with the appearance of virus-derived debris in the form of disrupted and isolated capsomeres. After treatment for 120 min no intact virus particles were observed. The action of wet (100 degrees C) or dry (60 degrees C) heat resulted in changes similar to those effected by UV radiation, with a rapid loss of viral outer capsid shell from the virions followed by stain penetration and disintegration of particles. Sodium hypochlorite, cetrimide and 70% ethanol induced a rapid loss of the outer capsid layer, but, compared with UV radiation or heat, a slower increase in the number of stain-penetrated particles was noted. This was particularly evident with cetrimide. Chlorhexidine and phenol had effects on virus structure only after extended periods of exposure, whilst glutaraldehyde treatment had little influence on virus morphology. Glutaraldehyde 2% v/v would appear to be most suitable for the disinfection of rotavirus-containing electronmicroscope grids before their examination.

Skin surface electron microscopy in Pityrosporum folliculitis. The role of follicular occlusion in disease and the response to oral ketoconazole.

The yeast Pityrosporum orbiculare is thought to cause the folliculitis associated with seborrheic eczema. However, a combination of mechanical and microbiological factors may be involved, with follicular occlusion leading to yeast overgrowth and folliculitis. Scanning electron microscopy was used to investigate this hypothesis. Skin biopsy specimens obtained from patients with Pityrosporum folliculitis were examined by scanning electron microscopy before and after oral ketoconazole therapy. Patients with active disease showed occlusion of noninflamed follicles, which resolved after ketoconazole treatment. Follicular occlusion was not present in biopsy specimens obtained from unaffected controls nor was it related to the presence of P orbiculare. These findings suggest that follicular occlusion may be a primary event in the development of this folliculitis, with yeast overgrowth a secondary occurrence. The beneficial effect of ketoconazole in this disease may be due to direct effects on the follicle.

Skin surface electron microscopy in Pityrosporum folliculitis. The role of follicular occlusion in disease and the response to oral ketoconazole.

The yeast Pityrosporum orbiculare is thought to cause the folliculitis associated with seborrheic eczema. However, a combination of mechanical and microbiological factors may be involved, with follicular occlusion leading to yeast overgrowth and folliculitis. Scanning electron microscopy was used to investigate this hypothesis. Skin biopsy specimens obtained from patients with Pityrosporum folliculitis were examined by scanning electron microscopy before and after oral ketoconazole therapy. Patients with active disease showed occlusion of noninflamed follicles, which resolved after ketoconazole treatment. Follicular occlusion was not present in biopsy specimens obtained from unaffected controls nor was it related to the presence of P orbiculare. These findings suggest that follicular occlusion may be a primary event in the development of this folliculitis, with yeast overgrowth a secondary occurrence. The beneficial effect of ketoconazole in this disease may be due to direct effects on the follicle.

Aetiology and outcome of severe community-acquired pneumonia.

Between January 1972 and December 1981, 50 patients with severe community-acquired pneumonia were admitted to the intensive care unit of a district general hospital. A causal pathogen was identified in 41 cases (82%). Streptococcus pneumoniae (16 cases), Legionella pneumophila (15 cases) and Staphylococcus aureus (5 cases) were the commonest. Assisted ventilation was required in 44 patients, of whom 25 died (57%). All 5 patients with staphylococcal pneumonia and 12(75%) with pneumococcal pneumonia died. Only 5 (33%) with Legionnaires' disease died. Mortality was significantly associated with age. Recommendations for the management of severe pneumonia are made.

Pathogenicity of natural and experimental listeriosis in newly hatched chicks.

The pathogenicity of Listeria monocytogenes for newly hatched chickens exposed to natural infection was examined. Organisms entered through the alimentary tract and dissemination followed bacteraemia. Among a number of symptoms recorded were unilateral and bilateral toe paralysis. In addition to gross abnormalities in the following tissues, histological lesions were seen in the liver, spleen, heart and kidneys of all infected chicks but brain lesions were observed only in birds with central nervous system involvement. The organism was recovered from some tissues derived from apparently healthy chicks as well as those with listeriosis. The use of trypsin in the isolation process increased the probability of a positive result from tissues, reduced the storage time needed and had no adverse effect on the rate of organism growth.