Faculty Bibliography

A concise, stereoselective, and convergent total synthesis of the unnatural enantiomer of the neodolastane diterpenoid heptemerone B has been completed. Saponification of (-)-heptemerone afforded (-)-guanacastepene E. The absolute stereochemistry of (-)-heptemerone B was thus established as 5-(S), the same as (-)-guanacastepene E. The longest linear sequence of the synthesis comprises 17 (18) steps from simple known starting materials. Our general synthetic approach integrates a diverse set of reactions, including an intramolecular Heck reaction to create one quaternary stereocenter and a cuprate conjugate addition for the establishment of the other. The central seven-membered ring was closed with an uncommon electrochemical oxidation, whereas the five-membered ring was formed through ring-closing metathesis. The absolute configuration of the two key building blocks was established through an asymmetric reduction and an asymmetric ene reaction.

Ion channels are gated by a variety of stimuli, including ligands, voltage, membrane tension, temperature, and even light. Natural gates can be altered and augmented using synthetic chemistry and molecular biology to develop channels with completely new functional properties. Light-sensitive channels are particularly attractive because optical manipulation offers a high degree of spatial and temporal control. Over the last few decades, several channels have been successfully rendered responsive to light, including the nicotinic acetylcholine receptor, gramicidin A, a voltage-gated potassium channel, an ionotropic glutamate receptor, alpha-hemolysin, and a mechanosensitive channel. Very recently, naturally occurring light-gated cation channels have been discovered. This review covers the molecular principles that guide the engineering of light-gated ion channels for applications in biology and medicine.

GFP (green fluorescent protein) fusion proteins have revolutionized research on protein dynamics at synapses. However, corresponding analyses usually involve protein expression methods that override endogenous regulatory mechanisms, and therefore cause overexpression and temporal or spatial misexpression of exogenous fusion proteins, which may seriously compromise the physiological validity of such experiments. These problems can be circumvented by using knock-in mutagenesis of the endogenous genomic locus to tag the protein of interest with a fluorescent protein. We generated knock-in mice expressing a fusion protein of the presynaptic active zone protein Munc13-1 and enhanced yellow fluorescent protein (EYFP) from the Munc13-1 locus. Munc13-1-EYFP-containing nerve cells and synapses are functionally identical to those of wild-type mice. However, their presynaptic active zones are distinctly fluorescent and readily amenable for imaging. We demonstrated the usefulness of these mice by studying the molecular dynamics of Munc13-1-EYFP at individual presynaptic sites. Fluorescence recovery after photobleaching (FRAP) experiments revealed that Munc13-1-EYFP is rapidly and continuously lost from and incorporated into active zones (tau1 approximately 3 min; tau2 approximately 80 min). Munc13-1-EYFP steady-state levels and exchange kinetics were not affected by proteasome inhibitors or acute synaptic stimulation, but exchange kinetics were reduced by chronic suppression of spontaneous activity. These experiments, performed in a minimally perturbed system, provide evidence that presynaptic active zones of mammalian CNS synapses are highly dynamic structures. They demonstrate the usefulness of the knock-in approach in general and of Munc13-1-EYFP knock-in mice in particular for imaging synaptic protein dynamics.

Chromatin remodeling and transcription regulation are tightly controlled under physiological conditions. It has been suggested that altered chromatin modulation and transcription dysfunction may play a role in the pathogenesis of Huntington's disease (HD). Increased histone methylation, a well established mechanism of gene silencing, results in transcriptional repression. ERG-associated protein with SET domain (ESET), a histone H3 (K9) methyltransferase, mediates histone methylation. We show that ESET expression is markedly increased in HD patients and in transgenic R6/2 HD mice. Similarly, the protein level of trimethylated histone H3 (K9) was also elevated in HD patients and in R6/2 mice. We further demonstrate that both specificity protein 1 (Sp1) and specificity protein 3 (Sp3) act as transcriptional activators of the ESET promoter in neurons and that mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic, interferes with the DNA binding of these Sp family transcription factors, suppressing basal ESET promoter activity in a dose dependent manner. The combined pharmacological treatment with mithramycin and cystamine down-regulates ESET gene expression and reduces hypertrimethylation of histone H3 (K9). This polytherapy significantly ameliorated the behavioral and neuropathological phenotype in the R6/2 mice and extended survival over 40%, well beyond any existing reported treatment in HD mice. Our data suggest that modulation of gene silencing mechanisms, through regulation of the ESET gene is important to neuronal survival and, as such, may be a promising treatment in HD patients.

The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation

Brain systems communicate by means of neuronal oscillations at multiple temporal and spatial scales. In anesthetized rats, we find that neocortical 'slow' oscillation engages neurons in prefrontal, somatosensory, entorhinal, and subicular cortices into synchronous transitions between UP and DOWN states, with a corresponding bimodal distribution of their membrane potential. The membrane potential of hippocampal granule cells and CA3 and CA1 pyramidal cells lacked bimodality, yet it was influenced by the slow oscillation in a region-specific manner. Furthermore, in both anesthetized and naturally sleeping rats, the cortical UP states resulted in increased activity of dentate and most CA1 neurons, as well as the highest probability of ripple events. Yet, the CA3-CA1 network could self-organize into gamma bursts and occasional ripples during the DOWN state. Thus, neo/paleocortical and hippocampal networks periodically reset, self-organize, and temporally coordinate their cell assemblies via the slow oscillation

The asymmetric total synthesis of (-)-bipinnatin J and its conversion into (+)-intricarene through a transannular 1,3-dipolar cycloaddition is described. In addition, the conversion of (-)-bipinnatin J into (+)-rubifolide and (+)-isoepilophodione B is reported. Biosynthetic relationships among furanocembranoids and the possible role of 1,3-dipolar cycloadditions in biosynthesis are discussed. [reaction: see text]