Flow cytometric quantitation of yeast a novel technique for use in animal model work and in vitro immunologic assays

Animal models of fungal and other infectious diseases often require that the number of organisms in tissue be quantified, traditionally by grinding organs, plating them on agar and counting colony forming units (CFU). This method is labor intensive, slow as some fungi require two weeks of culture and limited in reliability by poor plating efficiency. To circumvent these problems, we developed a flow cytometric method to quantify yeast. In vitro cultured Blastomyces dermatitidis, Cryptococcus neoformans, Candida albicans and Histoplasma capsulatum yeast were labelled with specific monoclonal or polyclonal antibodies to stain surface determinants or with Calcofluor to stain cell-wall chitin. A defined number of fluorescently labelled beads were added prior to acquisition by flow cytometry as a reference standard for quantitation. Beads were readily distinguished from yeast by forward scatter, side scatter and intensity of fluorescence. Cultured yeast were enumerated by both standard CFU determination and flow cytometry in a range of 10(2) to 10(7) cells. Only flow cytometry enabled discrimination of live and dead yeast by using appropriate fluorescent dyes. The flow cytometric method was applied to murine models of histoplasmosis and blastomycosis to quantify the burden of fungi in the lungs of infected mice. Labelling yeast with Calcofluor alone resulted in unacceptably high levels of nonspecific binding to mouse cell debris. In contrast, labelling H. capsulatum with a rabbit polyclonal antiserum and B. dermatitidis with a monoclonal antibody to the surface protein WI-1 permitted accurate quantitation. We conclude that this flow cytometry technique is rapid, efficient and reliable for quantifying the burden of infection in animal models of fungal disease. The technique also should lend itself to performing cytotoxicity assays that require discrimination of live and dead fungi, or phagocytosis assays that require discrimination of intracellular and extracellular organisms.     

Ventricular myxoma presenting as acute visual loss

We report a case of left ventricular myxoma with embolization to the left posterior cerebral artery, causing acute visual loss. The tumor was successfully resected and a follow-up echocardiography after 21 months revealed no evidence of tumor recurrence. The patient also had a past history of testicular seminoma. We believe that this is the first case report of an association of cardiac myxoma and testicular seminoma.     

Effect of the non-NMDA receptor antagonist GYKI 52466 on the microdialysate and tissue concentrations of amino acids following transient forebrain ischaemia

The effect of the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) on ischaemia-induced changes in the microdialysate and tissue concentrations of glutamate, aspartate, and gamma-aminobutyric acid (GABA) was studied in rats. Twenty minutes of four-vessel occlusion resulted in a transient increase in microdialysate levels of glutamate, aspartate, and GABA in striatum, cortex, and hippocampus. Administration of GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min intravenously starting 20 min before onset of ischaemia) inhibited ischaemia-induced increases in microdialysate glutamate and GABA in striatum without affecting the increases in hippocampus or cortex. Twenty minutes of four-vessel occlusion resulted in immediate small decreases and larger delayed (72 h) decreases in tissue levels of glutamate and aspartate. Transient increases in tissue levels of GABA were shown in all three structures at the end of the ischaemic period. At 72 h, after the ischaemic period, significantly reduced GABA levels were observed in striatum and hippocampus. GYKI 52466, given under identical conditions as above, augmented the ischaemia-induced decrease in striatal tissue levels of glutamate and aspartate, without significantly affecting the decreases in hippocampus and cortex. Twenty minutes of ischaemia resulted in a large increase in microdialysate dopamine in striatum. GYKI 52466 failed to inhibit this increase. Kainic acid (500 microM infused through the probe for 20 min) caused increases in microdialysate glutamate and aspartate in the striatum. GYKI 52466 (10 mg/kg bolus + 10 mg/kg/60 min) completely inhibited the kainic acid-induced glutamate release. In conclusion, the action of the non-NMDA antagonist, GYKI 52466, in the striatum is different from that in the cortex and hippocampus. The inhibition by GYKI 52466 of ischaemia-induced and kainate-induced increases in microdialysate glutamate concentration in the striatum may be related to the neuroprotection provided by GYKI 52466 in this region.     

Outcomes for antidepressant trials in late-life depression

Most randomized controlled trials of antidepressant efficacy in older depressive patients use outcome measures similar to those applied in younger patients. Defining subjects as geriatric based on chronological age alone misses the opportunity to study relationships among efficacy, safety, and the range of impairments associated with aging. Assessment of outcomes related to the medical comorbidities and disabilities associated with late-life depression is needed to understand the broad range of treatment effects. Assessment of changes in subjective and objective measures of functioning can address relationships between clinical state and health-related quality of life. Sensitive measurement of the number and severity of comorbid medical illnesses can precisely characterize the study sample and assess the impact of efficacious treatment on medical illnesses. In long-term studies of heterogeneous geriatric samples, use of appropriate outcome measures can determine the effect of efficacious treatment on changes in health status and functional independence.     

Trans activation of the Escherichia coli ato structural genes by a regulatory protein from Bacillus megaterium: potential use in polyhydroxyalkanoate production

A Bacillus megaterium genomic fragment, which encoded an activator homologous to sigma 54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B. megaterium screened for beta-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains.     

Marker selection for the transmission/disequilibrium test, in recently admixed populations

Various analytical, iterative and rebinning algorithms have been proposed for 3D reconstruction in positron emission tomography. This paper examines execution times of five analytical and rebinning algorithms. Meaningful comparisons are obtained by using similar software modules in all implementations. Reconstruction times are shown to differ by vast amounts: the Favor algorithm of Defrise et al is more than twice as fast as the widely used reprojection algorithm of Kinahan and Rogers; the Fourier rebinning algorithm (Fore) recently developed by Defrise is more than 15 times faster than the reprojection algorithm.     

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.