Prenatal in situ hybridization test for deleted steroid sulfatase gene

X-linked ichthyosis results from steroid sulfatase (STS) deficiency; 90% of affected patients have a complete deletion of the entire 146 kb STS gene on the distal X chromosome short arm (Xp22.3). In these families prenatal diagnosis and carrier testing can be completed in 2 days by hybridizing simultaneously 2 different cosmid probes labeled with fluorescein or Texas red and counterstaining interphase nuclear DNA with DAPI. An STS gene probe labeled with Texas red hybridizes specifically to the steroid sulfatase gene on the X chromosome. A second flanking probe labeled with fluorescein hybridizes to both the normal Y chromosome and normal and STS deleted X chromosomes. In this fashion the interphase nuclei of normal males, affected males, normal females, and carrier females can be distinguished unambiguously. Because normal males and carrier females each show two yellow-green fluorescein spots and one Texas red STS spot, use of this test prenatally requires determining fetal sex independently with repetitive X and Y chromosome-specific probes. This procedure can be used with lymphocytes, direct and cultured chorionic villus cells, direct and cultured amniocytes, and fibroblasts. Similar methods are anticipated to be useful for rapid diagnostic assessment of other aneuploid gene disorders.     

The correlation between two dietary assessments of carotenoid intake and plasma carotenoid concentrations: application of a carotenoid food-composition database

A newly available carotenoid food-composition database providing specific carotenoid values for > 2300 foods was linked to dietary data on 57 male nonsmokers to examine the association between dietary carotenoid intake and plasma carotenoid concentrations over 3 wk when free-living. Carotenoid intake was estimated from a food-frequency questionnaire (FFQ) and 7 d of food diaries with concurrent analysis of plasma carotenoid concentrations. After adjustment for energy intake, percentage of energy from alcohol, and plasma lipid concentrations, significant diet-plasma correlations for the FFQ and the food diaries (FD) included alpha-carotene (r = 0.29 and 0.43), beta-carotene (r = 0.36 FFQ only), beta-cryptoxanthin (r = 0.46 and 0.44), lutein (r = 0.44 FD only), and lycopene (r = 0.53 FD only). Dietary carotenoid intakes were associated with plasma carotenoid concentrations for all the carotenoids except for beta-carotene when food diaries were used whereas the diet-plasma correlation for the provitamin A carotenoids were consistently significant when the FFQ was used.     

The possible influence of the menstrual cycle on the adherence of Candida albicans to human buccal epithelial cells in vitro

Although the influence of the menstrual cycle on both vaginal candidosis and Candida albicans adherence to vaginal epithelial cells in vitro has been shown to be significant, similar studies have not been made on oral candidosis and adherence to buccal epithelial cells. The aim of this study was therefore to use an in vitro adherence assay to investigate the possible influence of the menstrual cycle on the adherence of C. albicans to buccal epithelial cells. Epithelial cells were collected from a single, healthy, female volunteer on days 5, 15, 22 and 28 of six menstrual cycles. Adherence of C. albicans was significantly higher to buccal epithelial cells collected on day 5 of the menstrual cycle when compared with days 15, 22 and 28, both in terms of the percentage of buccal epithelial cells with adherent C. albicans and the number of C. albicans adhering per 200 buccal epithelial cells in four out of six menstrual cycles (p < 0.001). This result indicates that hormonal influences should be considered when buccal epithelial cells are used in vitro to assess candidal adherence and may implicate hormonal factors in the aetiology of oral candidosis.     

Synthesis and evaluation of analogues of the partial agonist 6-(propyloxy)-4-(methoxymethyl)-beta-carboline-3-carboxylic acid ethyl ester (6-PBC) and the full agonist 6-(benzyloxy)-4-(methoxymethyl)-beta-carboline-3-carboxylic acid ethyl ester (Zk 93423) at wild type and recombinant GABAA receptors

A pharmacophore and an alignment rule have previously been reported for BzR agonist ligands. The design and synthesis of 6-(propyloxy)-4-(methoxymethyl)-beta-carboline-3-carboxylic acid ethyl ester (6-PBC, 24, IC50 = 8.1 nM) was based on this pharmacophore. When evaluated in vivo this ligand exhibited anticonvulsant/anxiolytic activity but was devoid of the muscle relaxant/ataxic effects of "classical" 1,4-benzodiazepines (i.e., diazepam). Significantly, 6-PBC 24 also reversed diazepam-induced muscle relaxation in mice. The 3-substituted analogues 40-46 and 48 of 6-PBC 24 and Zk 93423 27(IC50 = 1 nM) were synthesized and evaluated in vitro to determine what affect these modifications would have on the binding affinity at recombinant BzR subtypes. With the exception of the 3-amino ligands 40 and 41, all the beta-carbolines were found to exhibit high binding affinity at BzR sites. The 3-propyl ether derivative 45 was also evaluated in vivo and found to be devoid of any proconvulsant or anticonvulsant activity at doses up to 40 mg/kg. The 6-(1-naphthylmethyloxy) and 6-octyloxy analogues 25, 26, 28, and 29 of 6-PBC 24 were synthesized to further evaluate the proposed alignment of agonists vs inverse agonists in the pharmacophore of the BzR. In addition, ligands 26 and 29 were designed to probe the dimensions of lipophilic pocket L3 at the agonist site. The activity of 29 was evaluated in vivo; however, this analogue elicited no pharmacological effects at doses up to 80 mg/kg. These and other related beta-carbolines were also examined in five recombinant GABAA receptor subtypes. Ligands 52-61 all exhibited moderate to high affinity at GABAA receptors containing alpha1 subunits. These ligands will be useful in further defining the pharmacophore at alpha1 beta3 gamma2 receptors.     

Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis

The Gly93-->Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (approximately 50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.     

Interactions of inhibition and excitation in the light-evoked currents of X type retinal ganglion cells

The excitatory and inhibitory conductances driving the light-evoked currents (LECs) of cat and ferret ON- and OFF-center X ganglion cells were examined in sliced and isolated retina preparations using center spot stimulation in tetrodotoxin (TTX)-containing Ringer. ON-center X ganglion cells showed an increase in an excitatory conductance reversed positive to +20 mV during the spot stimulus. At spot offset, a transient inhibitory conductance was activated on many cells that reversed near ECl. OFF-center X ganglion cells showed increases in a sustained inhibitory conductance that reversed near ECl during spot stimulation. At spot offset, an excitatory conductance was activated that reversed positive to +20 mV. The light-evoked current kinetics of ON- and OFF-center X cells to spot stimulation did not significantly differ in form from their Y cell counterparts in TTX Ringer. When inhibition was blocked, current-voltage relations of the light-evoked excitatory postsynaptic currents (EPSCs) of both ON- and OFF-X cells were L-shaped and reversed near 0 mV. The EPSCs averaged between 300 and 500 pA at -80 mV. The metabotropic glutamate receptor agonist 2-amino-4-phosphonobutyric acid (APB), was used to block ON-center bipolar cell function. The LECs of ON-X ganglion cells were totally blocked in APB at all holding potentials. APB caused prominent reductions in the dark holding current and synaptic noise of ON-X cells. In contrast, the LECs of OFF-X ganglion cells remained in APB. An increase in the dark holding current was observed. The excitatory amino acid receptor antagonist combination of D-amino-5-phosphono-pentanoic acid (D-AP5) and 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxalinedione (NBQX) was used to block ionotropic glutamate receptor retinal neurotransmission. The LECs of all ON-X ganglion cells were totally blocked, and their holding currents were reduced similar to the actions of APB. For OFF-X ganglion cells, the antagonist combination always blocked the excitatory current at light-OFF; however, in many cells, the inhibitory current at light-ON remained. ON-center X ganglion cells receive active excitation during center illumination, and a transient inhibition at light-OFF. In contrast OFF-center X ganglion cells experience a sustained active inhibition during center illumination, and a shorter increase in excitation at light-offset. Cone bipolar cells provide a resting level of glutamate release on X ganglion cells on which their light-evoked currents are superimposed [corrected].