Activation of electrogenic sodium pump in hippocampal CA1 neurons following glutamate-induced depolarization

Intracellular recordings were obtained from guinea pig hippocampal CA1 pyramidal neurons maintained in vitro. Focal applications of glutamate produced depolarizations followed by prolonged hyperpolarizations. The mechanisms underlying this postglutamate hyperpolarization (PGH) were investigated. PGH did not reverse polarity with hyperpolarization to potentials at or near the presumed K+ equilibrium potential. A transient increase in conductance was associated with the PGH; control values returned well before the termination of PGH. Application of Mn2+, an antagonist of voltage-dependent calcium conductance, blocked synaptic transmission and the afterhyperpolarization (AHP) that follows a directly evoked train of action potentials but did not diminish the PGH or the transient conductance increase. Intracellular application of the calcium chelator ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid blocked AHP but did not affect PGH. Reductions in temperature from 37 to 27-32 degrees C reduced the amplitude of PGH and prolonged its duration but increased the amplitude and duration of AHP. The transient conductance increase associated with PGH was unaffected. Application of strophanthidin, a specific antagonist of Na+-K+-ATPase, reversibly blocked PGH and led to large increases in the amplitude and duration of the AHP. It is concluded that PGH is produced by activation of the electrogenic sodium pump by glutamate-induced excitation. As such, PGH is a useful physiological assay of electrogenic sodium transport. In addition, maintenance of the Na+ gradient by the sodium pump is important for the buffering of Ca2+ influx.     

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.     

Increased frequency of a new polymorphism in the cell division cycle 2 (cdc2) gene in patients with Alzheimer's disease and frontotemporal dementia

Recent studies show linkage between Alzheimer's disease (AD) and two loci on chromosome 10. The cell division cycle 2 (cdc2) gene is located close to one of the chromosome 10 markers, and is a candidate gene for AD since it is involved in the pathogenesis of AD. We sequenced coding exons and flanking intronic sequences and the promoter region on the cdc2 gene and found three new single nucleotide polymorphisms (SNPs). We analyzed 272 Caucasian AD cases, 160 controls and 70 cases with frontotemporal dementia (FTD) for these SNPs. Homozygosity for one of the SNPs (Ex6+7I/D) was more frequent in both AD and FTD cases than in controls. In the combined tauopathy (AD and FTD) group the odds ratio (OR) was 1.77 (95% CI 1.19-2.63) for the Ex6+7II genotype. Our findings suggest that the Ex6+7I allele is associated with tauopathies, both AD and FTD.     

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Frequency and species distribution of gliotoxin-producing Aspergillus isolates recovered from patients at a tertiary-care cancer center

Aspergillus isolates (n = 103) collected from cancer patients were screened to determine the taxonomic distribution and quantity of gliotoxin production. Gliotoxin was detected in 93% of Aspergillus fumigatus, 75% of A. niger, 25% of A. terreus, and 4% of A. flavus cultures. Gliotoxin concentrations were highest in cultures of A. fumigatus.     

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.