Faculty Bibliography

BACKGROUND: Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively. RESULTS: P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS: Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels

Clostridial botulinum neurotoxins (BoNTs) inhibit synaptic exocytosis; intoxication requires the di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol, colocalizing it with the substrate SNARE proteins. We investigate the dynamics of protein translocation across membranes using a sensitive single-molecule assay to track translocation events with millisecond resolution on lipid bilayers and on membrane patches of Neuro 2A cells. Translocation of BoNT/A LC by the HC is observed in real time as changes of channel conductance: the channel is occluded by the light chain during transit, and open after completion of translocation and release of cargo, acting intriguingly similar to the protein-conducting/translocating channels of the endoplasmic reticulum, mitochondria, and chloroplasts. Our findings support the notion of an interdependent, tight interplay between the HC transmembrane chaperone and the LC cargo that prevents LC aggregation and dictates the productive passage of cargo through the channel and completion of translocation. The protein-conducting channel of BoNT, a key element in the process of neurotoxicity, emerges therefore as a target for antidote discovery - a novel paradigm of paramount significance to health science and biodefense.

The mammalian brain is an enormously complex set of circuits composed of interconnected neuronal cell types. The analysis of central neural circuits will be greatly served by the ability to turn off specific neuronal cell types while recording from others in intact brains. Because drug delivery cannot be restricted to specific cell types, this can only be achieved by putting "silencer" transgenes under the control of neuron-specific promoters. Towards this end we have created a line of transgenic mice putting the Drosophila allatostatin (AL) neuropeptide receptor (AlstR) under the control of the tetO element, thus enabling its inducible expression when crossed to tet-transactivator lines. Mammals have no endogenous AL or AlstR, but activation of exogenously expressed AlstR in mammalian neurons leads to membrane hyperpolarization via endogenous G-protein-coupled inward rectifier K(+) channels, making the neurons much less likely to fire action potentials. Here we show that this tetO/AlstR line is capable of broadly expressing AlstR mRNA in principal neurons throughout the forebrain when crossed to a commercially-available transactivator line. We electrophysiologically characterize this cross in hippocampal slices, demonstrating that bath application of AL leads to hyperpolarization of CA1 pyramidal neurons, making them refractory to the induction of action potentials by injected current. Finally, we demonstrate the ability of AL application to silence the sound-evoked spiking responses of auditory cortical neurons in intact brains of AlstR/tetO transgenic mice. When crossed to other transactivator lines expressing in defined neuronal cell types, this AlstR/tetO line should prove a very useful tool for the analysis of intact central neural circuits.

OBJECTIVE: To determine the effects of smoking on eggs and subsequent embryo development by maternal exposure to cigarette smoke. DESIGN: Mice were exposed to cigarette smoke or cigarette smoke condensate (CSC) for 4 weeks and then examined for development and telomere function of embryos in vitro after fertilization. In addition, the effects of continuous smoke on embryo development and telomere length were determined by treating mice for 4 weeks, followed by continous exposure to cigarette smoke or CSC after fertilization. SETTING: Laboratory study. ANIMAL(S): CD1 mice. INTERVENTION(S): Mice were exposured to cigarette smoke or CSC. MAIN OUTCOME MEASURE(S): The percentage (rate) of blastocyst development, quality of embryos assessed by total cell number, apoptosis, Oct4 expression (a molecular marker of embryonic stem cells), telomere length and loss, and chromosomal instability were compared between smoke- and CSC- treated mice and sham-treated mice. RESULT(S): Mice exposed to cigarette smoke or CSC for 4 weeks exhibited increased egg fragmentation or delayed fertilization, thus reducing development to blastocysts in vitro. Fragmented eggs showed increased reactive oxygen species. Mice exposed to smoke or CSC showed increased apoptosis and altered expression of Oct4 in developed embryos. The effects of smoke or CSC on embryo development showed a dose-dependent relationship to exposure time. Exposure to smoke or CSC beginning 4 weeks before fertilzation altered expression of Oct4 and increased apoptosis in blastocysts. Notably, the rate of abnormal embryos significantly increased in the smoke and CSC groups. Smoke and CSC shortened telomeres in embryos, but their telomere shortening was not enough to induce major chromosome abnormalities in mice, which have unusually long telomeres. CONCLUSION(S): Together, the whole animal exposure model shows that cigarette smoke induces oxidative stress, telomere shortening, and apoptosis, and compromises embryo development in vivo

Considerable progress has recently been made in understanding the role of cardiolipin in mitochondria. In this brief review, we discuss new data that show how cardiolipin specifically contributes to the lateral organization of mitochondrial membranes. We argue that the function of cardiolipin has to be understood in the context of dynamic membrane assembly rather than static membrane structure, and we propose that remodeling of cardiolipin, i.e. the formation of uniformly substituted molecular species, may reduce the energy barrier of the assembly process

OBJECTIVES: To better categorize the epidemiologic profile, clinical features, and disease associations of loose anagen hair syndrome (LAHS) compared with other forms of childhood alopecia. DESIGN: Retrospective survey. SETTING: Academic pediatric dermatology practice. Patients Three hundred seventy-four patients with alopecia referred from July 1, 1997, to June 31, 2007. MAIN OUTCOME MEASURES: Epidemiologic data for all forms of alopecia were ascertained, such as sex, age at onset, age at the time of evaluation, and clinical diagnosis. Patients with LAHS were further studied by the recording of family history, disease associations, hair-pull test or biopsy results, hair color, laboratory test result abnormalities, initial treatment, and involvement of eyelashes, eyebrows, and nails. RESULTS: Approximately 10% of all children with alopecia had LAHS. The mean age (95% confidence interval) at onset differed between patients with LAHS (2.8 [1.2-4.3] years) vs patients without LAHS (7.1 [6.6-7.7] years) (P < .001), with 3 years being the most common age at onset for patients with LAHS. All but 1 of 37 patients with LAHS were female. The most common symptom reported was thin, sparse hair. Family histories were significant for LAHS (n = 1) and for alopecia areata (n = 3). In 32 of 33 patients, trichograms showed typical loose anagen hairs. Two children had underlying genetic syndromes. No associated laboratory test result abnormalities were noted among patients who underwent testing. CONCLUSIONS: Loose anagen hair syndrome is a common nonscarring alopecia in young girls with a history of sparse or fine hair. Before ordering extensive blood testing in young girls with diffusely thin hair, it is important to perform a hair-pull test, as a trichogram can be instrumental in the confirmation of a diagnosis of LAHS

Armadillo family proteins known as plakophilins have been characterized as structural components of desmosomes that stabilize and strengthen adhesion by enhancing attachments with the intermediate filament cytoskeleton. However, plakophilins and their close relatives are emerging as versatile scaffolds for multiple signaling and metabolic processes that not only facilitate junction dynamics but also more globally regulate diverse cellular activities. While perturbation of plakophilin functions contribute to inherited diseases and cancer pathogenesis, the functional significance of the multiple PKP isoforms and the mechanisms by which their behaviors are regulated remain to be elucidated

The anterior cingulate cortex (ACC) is a component of the limbic system implicated in a wide variety of functions spanning motor and sensory information processing, memory, attention, novelty detection, and comparisons of expectation versus outcome. It remains unclear how much of this functional diversity stems from differences in methodology or interpretation versus truly reflecting the range of processes in which the ACC is involved. In the present study, ACC neuronal activity was examined in freely behaving mice (C57BL6/J) under conditions allowing investigation of many of the cited functions in conditions free from externally applied rules: tests of novel object and novel location recognition memory. Behavioral activity and neuronal activity were recorded first in the open field, during the initial exposure and subsequent familiarization to two identical objects, and finally during the recognition memory tests. No discernible stable firing correlates of ACC neurons were found in the open field, but the addition of objects led to lasting changes in the firing patterns of many ACC neurons around one or both of the object locations. During the novel location test, some neurons followed the familiar object to its new location, others fired exclusively where the object had been, and yet others fired to both current and former object locations. Many of these same features were observed during tests of object recognition memory. However, the magnitude of the neuronal preference for the novel or the familiar object was markedly greater than that observed during either the tests of location recognition or novel object preferences in animals that did not exhibit the expected behavior. The present study reveals, for the first time, single-neuron correlates of object and location recognition behaviors in the rodent ACC and suggests that neurons of the ACC provide a distributed representation of all of the salient features of a task.

OBJECTIVE: To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. METHODS: 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. RESULTS: After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. CONCLUSION: The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

Mitochondrial dysfunction, oxidative stress and reductions in thiamine-dependent enzymes have been implicated in multiple neurological disorders including Alzheimer's disease (AD). Experimental thiamine deficiency (TD) is an established model for reducing the activities of thiamine-dependent enzymes in brain. TD diminishes thiamine-dependent enzymes throughout the brain, but produces a time-dependent selective neuronal loss, glial activation, inflammation, abnormalities in oxidative metabolism and clusters of degenerating neurites in only specific thalamic regions. The present studies tested how TD alters brain pathology in Tg19959 transgenic mice over expressing a double mutant form of the amyloid precursor protein (APP). TD exacerbated amyloid plaque pathology in transgenic mice and enlarged the area occupied by plaques in cortex, hippocampus and thalamus by 50%, 200% and 200%, respectively. TD increased Abeta(1-42) levels by about three fold, beta-CTF (C99) levels by 33% and beta-secretase (BACE1) protein levels by 43%. TD-induced inflammation in areas of plaque formation. Thus, the induction of mild impairment of oxidative metabolism, oxidative stress and inflammation induced by TD alters metabolism of APP and/or Abeta and promotes accumulation of plaques independent of neuron loss or neuritic clusters