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Human embryonic stem (hES) cells are defined by their extensive self-renewal capacity and their potential to differentiate into any cell type of the human body. The challenge in using hES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential toward the derivation of a specific cell fate. Within the nervous system, hES cells have been shown to differentiate in vitro into neural progenitor cells, neurons, and astrocytes. However, to our knowledge, the selective derivation of any given neuron subtype has not yet been demonstrated. Here, we describe conditions to direct hES cells into neurons of midbrain dopaminergic identity. Neuroectodermal differentiation was triggered on stromal feeder cells followed by regional specification by means of the sequential application of defined patterning molecules that direct in vivo midbrain development. Progression toward a midbrain dopamine (DA) neuron fate was monitored by the sequential expression of key transcription factors, including Pax2, Pax5, and engrailed-1 (En1), measurements of DA release, the presence of tetrodotoxin-sensitive action potentials, and the electron-microscopic visualization of tyrosinehydroxylase-positive synaptic terminals. High-yield DA neuron derivation was confirmed from three independent hES and two monkey embryonic stem cell lines. The availability of unlimited numbers of midbrain DA neurons is a first step toward exploring the potential of hES cells in preclinical models of Parkinson's disease. This experimental system also provides a powerful tool to probe the molecular mechanisms that control the development and function of human midbrain DA neurons.

Based on the hypothesis that genetic modification of freshly isolated alveolar macrophages (AM) with the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA would induce AM to proliferate, this study focuses on the ability of adenoviral (Ad) vectors to transfer and efficiently express the murine (m) GM-CSF cDNA in murine AM with consequent expansion in the number of AM in vitro and in vivo. To demonstrate that an Ad vector can effectively transfer and express genes in AM, murine AM recovered by bronchoalveolar lavage from the lung of Balb/c mice were infected with an Ad vector coding for green fluorescent protein (GFP) in vitro and expressed GFP in a dose-dependent fashion. Infection of AM with an Ad vector containing an expression cassette coding for mGM-CSF led to GM-CSF expression and to AM proliferation in vitro. When AM infected with AdGFP were returned to the respiratory tract of syngeneic recipient mice, GFP-expressing cells could still be recovered by bronchoalveolar lavage 2 weeks later. In vitro infection of AM with AdmGM-CSF and subsequent transplantation of the genetically modified AM to the lungs of syngeneic recipients led to GM-CSF expression in vivo. Strikingly, the AM recovered by lavage 5 weeks after transplantation demonstrated an increased rate of proliferation, and the total number of alveolar macrophages was 1.9-fold greater than controls. Importantly, the increase in the numbers of AM was selective (ie, other inflammatory cell numbers were unchanged), and there was no modification to the lung architecture. Thus, it is feasible to genetically modify AM with Ad vectors and to use this strategy to modify the behavior of AM in vivo. Based on the importance of AM in the primary defense of the respiratory epithelial surface, this strategy may be useful in enhancing pulmonary defenses in immunodeficiency states.

Functional and organic alterations such as sclerosis of the internal anal sphincter muscle are often discussed as a cause of proctologic diseases. Systematic histopathologic examinations of this muscle are rare, although the internal anal sphincter is of primary significance for the continence of the anal canal and its functional efficiency. The authors believe that the degree of formation of connective tissue in this unstriated muscle depends on the age of the examined specimen. The authors can demonstrate that sclerosis is a physiologic process of aging.

Propofol (2,6-diisopropylphenol) is a widely used intravenous general anesthetic, which has been reported to produce bradycardia in patients at concentrations associated with profound sedation and loss of consciousness. Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels conduct a monovalent cationic current I(h) (also known as I(q) or I(f)) that contributes to autorhythmicity in both the brain and heart. Here we studied the effects of propofol on recombinant HCN1, HCN2, and HCN4 channels and found that the drug inhibits and slows activation of all three channels at clinically relevant concentrations. In oocyte expression studies, HCN1 channel activation was most sensitive to slowing by propofol (EC(50) values of 5.6 +/- 1.0 microM for fast component and 31.5 +/- 7.5 microM for slow component). HCN1 channels also showed a marked propofol-induced hyperpolarizing shift in the voltage dependence of activation (EC(50) of 6.7 +/- 1.0 microM) and accelerated deactivation (EC(50) of 4.5 +/- 0.9 microM). Furthermore, propofol reduced heart rate in an isolated guinea pig heart preparation over the same range of concentrations. These data suggest that propofol modulation of HCN channel gating is an important molecular mechanism that can contribute to the depression of central nervous system function and also lead to bradyarrhythmias in patients receiving propofol during surgical anesthesia.

Background Volatile anesthetics prolong inhibitory postsynaptic potentials in central neurons an allosteric action on the gamma-aminobutyric acid type A (GABA(A)) receptor, an effect that may underlie the hypnotic actions of these agents. Inhaled anesthetics such as isoflurane act to enhance responses to submaximal concentrations of GABA, but it is not clear whether their effect is mediated by an increase in the binding of the agonist or by changes in receptor gating behavior. To address this question, the authors studied the effects of isoflurane on a mutant GABA(A) receptor with a gating defect that decreases receptor sensitivity by lowering agonist efficacy. They then compared the effects of clinically relevant concentrations of isoflurane on the actions of GABA and piperidine-4-sulfonic acid (P4S), a partial agonist at the GABA(A) receptor. Methods The authors created a mutant of the GABA receptor alpha subunit (L277A) by site-directed mutagenesis. The mutant subunit was coexpressed with beta(2) and gamma(2S) subunits in HEK293 cells, and responses to GABA and P4S were recorded using the whole-cell patch clamp technique. EC values were determined for the full agonist GABA and the partial agonist P4S. The authors also determined the relative efficacy (epsilon) of P4S. These measurements were then repeated in the presence of isoflurane. Results The concentration-response curve for GABA was shifted to the right (EC(50) = 278 microm) in the alpha(1)(L277A)beta(2)gamma(2S) mutant receptor, compared with the corresponding wild-type alpha(1)beta(2)gamma(2S) GABA(A) receptor (EC(50) = 16 microm). P4S is a partial agonist at both receptors, with a dramatically decreased relative efficacy at the mutant receptor (epsilon = 0.24). When the mutant receptor was studied in the presence of isoflurane, the concentration-response curves for both GABA and P4S were shifted to the left (EC(50) for GABA = 78 microm); the efficacy of P4S also increased significantly (epsilon = 0.40). Conclusion By studying a mutant GABA receptor with impaired gating, the authors were able to demonstrate clearly that isoflurane can increase the efficacy of a partial agonist, as well as increase agonist potency. These data suggest that the volatile anesthetic isoflurane exerts at least some of its effects on the GABA(A) receptor via alterations in gating rather than simply changing binding or unbinding of the agonist.