Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis - acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.      

Abnormalities of interleukin 2 receptor expression associated with decreased antigen-induced lymphocyte proliferation in patients with AIDS and related disorders.

Several parameters of lymphocyte interleukin 2 receptor (IL-2R) expression in response to tetanus toxoid (TT) were monitored using a receptor-specific monoclonal antibody and flow cytometry in patients with acquired immunodeficiency syndrome (AIDS, n = 6), AIDS-related complex (ARC, n = 8), or lymphadenopathy syndrome (LAS, n = 9). These parameters, measured on days 6 and 7 of culture, included (a) the percentage of recovered lymphocytes expressing IL-2R, (b) the number of IL-2R-positive cells/lymphocyte trigger region (a relative measure of the number of IL-2R-positive cells per culture), and (c) mean fluorescence intensity of IL-2R-positive cells. Data were then assessed in relation to DNA synthesis on day 7 of culture. Normal DNA synthesis was observed in nine patients (Pt-N group), and the mean values for all three IL-2R expression parameters in this group did not significantly differ from those of the healthy control group (n = 20). In contrast, 14 patients exhibited low proliferation (Pt-L group) and mean values for all three parameters were significantly lower in this group compared to both the Pt-N and control groups. When considering individual patient values, day 7 IL-2R+ cell number proved the best predictor of DNA synthesis results: all 14 patients in the Pt-L group exhibited low IL-2R+ cell number, while seven of nine patients in the Pt-N group exhibited normal IL-2R+ cell number. Thus, low TT-induced lymphocyte proliferation in AIDS and related disorders is associated with defective IL-2R expression. Further, assessment of IL-2R+ cell number appears to be a valid alternative assay for monitored antigen-induced T cell proliferation in vitro.     

Membrane permeability changes during stimulation of isolated salivary glands of Calliphora by 5-hydroxytryptamine.

1. The membrane resistance of isolated salivary glands was found to decrease in response to 5-HT. The change in resistance was calcium-dependent. 2. The resistance change of the apical membrane was found to be much greater than the change in resistance of the basal membrane. 3. Potential responses under current-clamped conditions showed that one part of the biphasic response to 5-HT (attributed to an increase in chloride permeability) could be reversed and the other part (attributed to an increase in a potassium pump) could not. 4. These observations have been incorporated into a model which, on evaluation, predicts all of the observed potential changes during the action of 5-HT. It suggests that the potential responses reflect changes in the internal chloride concentration produced by the calcium-dependent increases in chloride permeability.     

Electrophysiology of dentate gyrus granule cells

The orthodromic synaptic responses, membrane properties, and responses of dentate gyrus granule cells (DGCs) to several convulsant agents were studied in the in vitro hippocampal slice preparation. Orthodromic stimulation via the perforant pathway (PP) evoked excitatory-inhibitory postsynaptic potentials (EPSP-IPSP) sequences in 27 of 34 DGCs studied. In the majority, only one action potential could be evoked by supramaximal orthodromic stimulation. In recordings from DGC somata, overshooting spikes could be evoked either orthodromically or by current injections. Small-amplitude, fast transients were seen in 5 of 34 DGCs. The current/voltage (I-V) characteristic of most DGCs was linear throughout a range of membrane potentials between 15 and 20 mV negative and 5 and 15 mV positive to the resting potential. At the extremes of this range nonohmic behavior was noted. Exposure of slices to agents that block IPSPs, such as penicillin, bicuculline, picrotoxin, and media containing low Cl- concentrations, eliminated PP-evoked hyperpolarizations in DGCs and prolonged the repolarizing phase of the PP EPSP. In contrast to findings in hippocampal pyramidal cells and neocortical neurons, blockade of IPSPs did not lead to the development of orthodromically evoked slow depolarizations and burst discharges. After slices were exposed to 5 mM tetraethylammonium, current pulses evoked slow spikes, which were resistant to tetrodotoxin and presumably mediated by Ca2+. Spontaneous burst discharges or bursts evoked by brief depolarizing pulses did not occur under these conditions. Substitution of Ba2+ for Ca2+ in the perfusion solution resulted in development of spontaneous slow membrane depolarizations and burst discharges in DGCs. Burst discharges could be directly evoked and spikes were prolonged and resistant to tetrodotoxin (TTX). After hyperpolarizations lasting 200-1,000 ms, associated with a conductance increase and presumably due to a Ca2+-activated K+ conductance, followed directly evoked spike trains in 5 of 20 DGCs. These data suggest that Ca2+ conductances may be evoked in DGCs under certain circumstances but are not prominent during activation of DGCs under standard in vitro recording conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro activation of T lymphocytes from HIV-seropositive blood donors. II. Decreased mitogen-induced expression of interleukin 2 receptor by both CD4 and CD8 cell subsets

Mononuclear cells (MC) from many individuals exposed to human immunodeficiency virus (HIV) exhibit a reduced proliferative response to a suboptimal concentration of phytohemagglutinin (PHA). However, the relative contributions of the 2 major T-cell subsets, namely CD4 and CD8 lymphocytes, to this reduced response remain unclear. Based on reports that interleukin 2 receptor (IL2R) expression correlates well with proliferative responses in HIV infection, we used dual-color cytofluorometry to measure IL2R expression by CD4 and CD8 cells following PHA activation of MC from HIV-seropositive blood donors. For data analysis, this study group was divided into two subgroups on the basis of DNA synthesis responses (seropositive with normal DNA synthesis, designated sero + NML, or seropositive with decreased DNA synthesis, designated sero + LOW). When compared to the seronegative control and sero + NML groups, the sero + LOW group exhibited significant reductions in the percentage of MC expressing IL2R, the proportion of CD4 cells expressing IL2R, and the proportion of CD8 cells expressing IL2R. In contrast, these parameters were unchanged in the sero + NML group compared to the control group. These findings show that reduced PHA-induced proliferative responses by MC from HIV-infected persons are associated with decreased IL2R expression by both CD4 and CD8 lymphocyte subsets.     

Effect of exogenous insulin on meal patterns and stomach emptying in the spiny mouse

Male adult spiny mice (Acomys cahirinus) were acutely challenged with a single dose of regular insulin or saline vehicle SC; either food intake, meal frequency and meal duration, or stomach emptying were then measured. Meal frequency, as well as amount eaten, was significantly higher over a 6-hr period following both 10 and 30 U/kg of insulin than following vehicle injection. Meal duration remained essentially the same across all conditions. When 30 U/kg of insulin was administered either 15 min prior to, or immediately after, a solid food meal, stomach emptying (as measured by dry weight of recovered stomach contents) was accelerated relative to vehicle controls. These data are generally consistent with and extend the comparative literature suggesting a possible link between rate of stomach emptying and insulin-induced hyperphagia in some species.     

The cotton rat: an underutilized animal model for human infectious diseases can now be exploited using specific reagents to cytokines, chemokines, and interferons.

The cotton rat represents the best or only animal model for a large number of human infectious diseases, and it may be unique among small laboratory animals in its susceptibility to several potential agents of bioterrorism. Although the cotton rat is a reliable model to define pathologic changes produced during infection with human pathogens, the lack of specific reagents has precluded a more extensive analysis of the molecular basis of pathogenesis. Here, we report the cloning of 24 cotton rat genes encoding various cytokines, chemokines, and interferons (IFNs). Analysis of the expression of most of these genes was performed by RT-PCR in cotton rat macrophages during treatment with lipopolysaccharide (LPS) and in cotton rat lungs after infection with influenza virus. The availability of these reagents will provide the tools for molecular analysis of pathogenesis and immune responses to a wide variety of pathogens and set the basis for the development of new prophylactic and therapeutic strategies against human infectious diseases.     

Relative contributions of passive equilibrium and active transport to the distribution of chloride in mammalian cortical neurons

1. Active and passive factors affecting the chloride gradient of cortical neurons were assessed using intracellular recordings from neurons in slices of cingulate cortex maintained in vitro. The chloride equilibrium potential (ECl-) was estimated indirectly from the reversal potentials of responses to perisomatic gamma-aminobutyric acid (GABA) application and the Cl(-)-dependent inhibitory postsynaptic potential (IPSP). Under control conditions the mean resting potential (Vm; -69.7 mV) was not significantly different than the mean IPSP reversal potential (EIPSP; -70.1 mV). 2. Increasing the external potassium concentration ([K+]o) from 1 to 10 mM shifted the mean EIPSP from -80.4 to -61.8 mV. The mean EIPSP was approximately equal to the mean Vm at all [K+]oS. The conditions of Donnan equilibrium are not met in [K+]o less than 10 mM. 3. Polarization of Vm up to 20 mV away from EIPSP for 4 min with maintained current injection had no significant effect on EIPSP. 4. The GABA reversal potential was maintained 37-52 mV less negative than Vm after equilibration in saline in which the external chloride concentration had been reduced from 133 to 5 mM by substitution with isethionate. Vm and input resistance were not significantly different from control values in cells recorded under these conditions. 5. We conclude that Cl- is not passively distributed in cortical neurons, perhaps due to a low resting Cl- permeability. 6. Impalement with electrodes containing 2 M KCl resulted in a rapid 10 mV depolarizing shift in EIPSP that then remained relatively constant. Intracellular iontophoresis of Cl- resulted in a further depolarizing shift of EIPSP of 5-10 mV that returned to control in less than 1 min. The time course of recovery of IPSP amplitude could be fit with a single exponential having a mean time constant of 6.9 +/- 1.5 s and was independent of the amount of Cl- injected or stimulation frequency. 7. Reductions in temperature from 37 to 32 degrees C significantly increased the mean time constant of IPSP recovery from Cl- injection to 11.1 +/- 3.3 s, corresponding to Q10 = 2.6.(ABSTRACT TRUNCATED AT 400 WORDS)