Lambert-Eaton sera reduce low-voltage and high-voltage activated Ca2+ currents in murine dorsal root ganglion neurons.

Voltage-gated Ca2+ channels are categorized as either high-voltage activated (HVA) or low-voltage activated (LVA), and a subtype (or subtypes) of HVA Ca2+ channels link the presynaptic depolarization to rapid neuro-transmitter release. Reductions in transmitter release are characteristic of the autoimmune disorder, Lambert-Eaton syndrome (LES). Because antibodies from LES patients reduce Ca2+ influx in a variety of cell types and disrupt the intramembrane organization of active zones at neuromuscular synapses, specificity of LES antibodies for the Ca2+ channels that control transmitter release has been suggested as the mechanism for disease. We tested sera from four patients with LES. Serum samples from three of the four patients reduced both the maximal LVA and HVA Ca2+ conductances in murine dorsal root ganglion neurons. Thus, even though LES is expressed as a neuromuscular and autonomic disorder, our studies suggest that Ca2+ channels may be broadly affected in LES patients. To account for the specificity of disease expression, we suggest that incapacitation of only a fraction of the Ca2+ channels clustered at active zones would severely depress transmitter release. In particular, if several Ca2+ channels in a cluster are normally required to open simultaneously before transmitter release becomes likely, the loss of a few active zone Ca2+ channels would exponentially reduce the probability of transmitter release. This model may explain why LES is expressed as a neuromuscular disorder and can account for a clinical hallmark of LES, facilitation of neuromuscular transmission produced by vigorous voluntary effort.

Antisense oligonucleotides against rat brain α1E DNA and its atrial homologue decrease T-type calcium current in atrial myocytes

Low voltage-activated, or T-type, calcium currents are important regulators of neuronal and muscle excitability, secretion, and possibly cell growth and differentiation. The gene (or genes) coding for the pore-forming subunit of low voltage-activated channel proteins has not been unequivocally identified. We have used reverse transcription–PCR to identify partial clones from rat atrial myocytes that share high homology with a member of the E class of calcium channel genes. Antisense oligonucleotides targeting one of these partial clones (raE1) specifically block the increase in T-current density that normally results when atrial myocytes are treated with insulin-like growth factor 1 (IGF-1). Antisense oligonucleotides targeting portions of the neuronal rat α1E sequence, which are not part of the clones detected in atrial tissue, also block the IGF-1-induced increase in T-current, suggesting that the high homology to α1E seen in the partial clone may be present in the complete atrial sequence. The basal T-current expressed in these cells is also blocked by antisense oligonucleotides, which is consistent with the notion that IGF-1 up-regulates the same gene that encodes the basal current. These results support the hypothesis that a member of the E class of calcium channel genes encodes a low voltage-activated calcium channel in atrial myocytes.

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 μg of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes

We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.

Modeling stochastic gene expression: Implications for haploinsufficiency

There is increasing recognition that stochastic processes regulate highly predictable patterns of gene expression in developing organisms, but the implications of stochastic gene expression for understanding haploinsufficiency remain largely unexplored. We have used simulations of stochastic gene expression to illustrate that gene copy number and expression deactivation rates are important variables in achieving predictable outcomes. In gene expression systems with non-zero expression deactivation rates, diploid systems had a higher probability of uninterrupted gene expression than haploid systems and were more successful at maintaining gene product above a very low threshold. Systems with relatively rapid expression deactivation rates (unstable gene expression) had more predictable responses to a gradient of inducer than systems with slow or zero expression deactivation rates (stable gene expression), and diploid systems were more predictable than haploid, with or without dosage compensation. We suggest that null mutations of a single allele in a diploid organism could decrease the probability of gene expression and present the hypothesis that some haploinsufficiency syndromes might result from an increased susceptibility to stochastic delays of gene initiation or interruptions of gene expression.

X-ray emission from clusters and groups of galaxies

Recent major advances in x-ray imaging and spectroscopy of clusters have allowed the determination of their mass and mass profile out to ≈1/2 the virial radius. In rich clusters, most of the baryonic mass is in the gas phase, and the ratio of mass in gas/stars varies by a factor of 2–4. The baryonic fractions vary by a factor of ≈3 from cluster to cluster and almost always exceed 0.09 h50−[3/2] and thus are in fundamental conflict with the assumption of Ω = 1 and the results of big bang nucleosynthesis. The derived Fe abundances are 0.2–0.45 solar, and the abundances of O and Si for low redshift systems are 0.6–1.0 solar. This distribution is consistent with an origin in pure type II supernova. The amount of light and energy produced by these supernovae is very large, indicating their importance in influencing the formation of clusters and galaxies. The lack of evolution of Fe to a redshift of z ≈ 0.4 argues for very early enrichment of the cluster gas. Groups show a wide range of abundances, 0.1–0.5 solar. The results of an x-ray survey indicate that the contribution of groups to the mass density of the universe is likely to be larger than 0.1 h50−2. Many of the very poor groups have large x-ray halos and are filled with small galaxies whose velocity dispersion is a good match to the x-ray temperatures.

Low ethanol concentrations enhance GABAergic inhibitory postsynaptic potentials in hippocampal pyramidal neurons only after block of GABAB receptors.

Despite considerable evidence that ethanol can enhance chloride flux through the gamma-aminobutyric acid type A (GABA/A/) receptor-channel complex in several central neuron types, the effect of ethanol on hippocampal GABAergic systems is still controversial. Therefore, we have reevaluated this interaction in hippocampal pyramidal neurons subjected to local monosynaptic activation combined with pharmacological isolation of the various components of excitatory and inhibitory synaptic potentials, using intracellular current- and voltage-clamp recording methods in the hippocampal slice. In accord with our previous findings, we found that ethanol had little effect on compound inhibitory postsynaptic potentials/currents (IPSP/Cs) containing both GABA/A/ and GABA/B/ components. However, after selective pharmacological blockade of the GABA/B/ component of the IPSP (GABA/B/-IPSP/C) by CGP-35348, low concentrations of ethanol (22-66 mM) markedly enhanced the peak amplitude, and especially the area, of the GABA/A/ component (GABA/A/-IPSP/C) in most CA1 pyramidal neurons. Ethanol had no significant effect on the peak amplitude or area of the pharmacologically isolated GABA/B/-inhibitory postsynaptic current (IPSC). These results provide new data showing that activation of GABAB receptors can obscure ethanol enhancement of GABA/A/ receptor function in hippocampus and suggest that similar methods of pharmacological isolation might be applied to other brain regions showing negative or mixed ethanol-GABA interactions.