GeneID in Drosophila

GeneID is a program to predict genes in anonymous genomic sequences designed with a hierarchical structure. In the first step, splice sites, and start and stop codons are predicted and scored along the sequence using position weight matrices (PWMs). In the second step, exons are built from the sites. Exons are scored as the sum of the scores of the defining sites, plus the log-likelihood ratio of a Markov model for coding DNA. In the last step, from the set of predicted exons, the gene structure is assembled, maximizing the sum of the scores of the assembled exons. In this paper we describe the obtention of PWMs for sites, and the Markov model of coding DNA in Drosophila melanogaster. We also compare other models of coding DNA with the Markov model. Finally, we present and discuss the results obtained when GeneID is used to predict genes in the Adh region. These results show that the accuracy of GeneID predictions compares currently with that of other existing tools but that GeneID is likely to be more efficient in terms of speed and memory usage.     

Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections

The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings, one aqueous sample, and one vitreous sample; one of them was negative by culture. Molecular fungal identification was successful in all cases. Bacterial detection by PCR was positive for three aqueous samples and one vitreous sample; one of these was negative by culture. Amplification of ITS2/5.8S rDNA and molecular typing shows potential as a rapid technique for identifying fungi in ocular samples.     

Aspects of the Central Integration of Arterial Baroreceptor and Cardiac Ventricular Receptor Reflexes in the Cat

The possible central integrative mechanisms, responsible for the earlier reported, differentiated reflex engagement of the renal and muscle vessels and the heart from cardiac ventricular receptors and arterial baroreceptors, respectively, were analyzed in atropinized cats. The reflex renal vessel, muscle vessel and heart rate responses, expressed as per cent of maximum, to graded activations of arterial baroreceptors (sinus pressure variations) and stimulations of ventricular receptor afferents in the cardiac nerve were systematically compared. Cardiac nerve stimulation with low frequencies was found to elicit more pronounced reflex renal vessel responses than muscle vessel and heart rate responses. In contrast, elevations of sinus pressure induced equally pronounced renal and muscle vessel responses. High frequency cardiac nerve stimulation elicited maximal reflex renal vessel responses, but only submaximal effects on muscle vessels and heart rate, while intense baroreceptor stimulation induced maximal reflex effector responses throughout. The submaximal heart rate response to cardiac nerve stimulation is probably due to a simultaneous activation of excitatory afferents. On the other hand, the less pronounced muscle than renal vessel responses when the cardiac nerve was stimulated probably reflect a relatively sparse innervation of muscle vasomotor neurons by ventricular receptor afferents, which seem instead to be preferentially oriented towards renal vasomotor and, possibly, cardiac motor neurons.     

Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract

BACKGROUND: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. OBJECTIVE: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. METHODS: This was a randomized, double-blind, placebo-controlled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebo-controlled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebo-controlled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG(4), and serum cytokines before and after treatment. RESULTS: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P = .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG(4) and IL-10 levels after immunotherapy in only the active group. CONCLUSION: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment.     

The prion protein in human neuromuscular diseases

The basis of human prion diseases affecting the nervous system is accumulation of a disease-associated conformer (PrPSc) of the normal cellular prion protein (PrPC). Earlier studies demonstrated increased expression of PrPC in inclusion body myositis (IBM), dermato-, and polymyositis, as well as neurogenic muscle atrophy. To define the spectrum and reliability of PrPC immunoreactivity, its expression was examined systematically in a series of pathologically characterized muscular disorders by means of immunohistochemistry, confocal laser microscopy, and immunogold electron microscopy. Anti-PrPC immunolabelling of rimmed vacuoles was observed in IBM, inclusions of myofibrillary myopathy, targets, regenerating, and atrophic fibres, mononuclear cells, in addition to ragged red fibres in mitochondrial myopathies, and focal sarcolemmal immunostaining in non-diseased controls. Quantitative analysis demonstrated that, in neurogenic muscle lesions, anti-PrPC staining detects a significantly broader spectrum of fibres than anti-vimentin or anti-NCAM. In dystrophic muscle, PrPC expression was mainly restricted to regenerating fibres. In IBM, PrPC expression was not confined to rimmed vacuoles or vacuolated fibres and only a small percentage (7.1%) of rimmed vacuoles were PrPC positive. Ultrastructurally, PrPC was observed in the cytoplasm of lymphocytes, in the myofibrillar network of targets, and in rimmed vacuoles. Knowledge of disease circumstances with altered expression of PrPC is important in the setting of a potentially increased chance for extraneural PrPC-PrPSc conversion. In addition, our observations suggest that PrPC may have a general stress-response effect in various neuromuscular disorders.     

A long lasting Ca2+ -activated outward current in guinea-pig atrial myocytes

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region –15 to +40mV. This hump was reversibly suppressed by Co²⁺ (3 mM) or nitrendipine (5 M) and enhanced by Bay K 8644 (5 M). The maintained outward current component suppressed by Co²⁺ extended between –15.2±1.9 mV and +39.5 ±1.7 mV (mean±SEM of 14 cells) and has an amplitude of 95.7±9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co²⁺ -sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K⁺ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 M), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 M) or quinine (100 M) but not TEA (5–50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca²⁺ -activated K⁺ currents in heart cells since they also inhibited the L-Type Ca²⁺ current. It is concluded that a long lasting Ca²⁺ -activated outward current, probably mainly carried by K⁺ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.