Faculty Bibliography

BACKGROUND: Sodium channel alpha-subunits in ventricular myocytes (VMs) segregate either to the intercalated disc, or to lateral membranes, where they associate with region-specific molecules. OBJECTIVE: To determine the functional properties of sodium channels as a function of their location in the cell. METHODS: Local sodium currents were recorded from adult rodent VMs and Purkinje cells using the cell-attached macropatch configuration. Electrodes were placed either in the cell midsection (M), or cell end (area originally occupied by the intercalated disc; ID). Channels were identified as TTX-sensitive (TTX-S) or TTX-resistant (TTX-R) by application of 100 nM TTX. RESULTS: Average peak-current amplitude was larger in ID than M, and largest at site of contact between attached cells. TTX-S channels were found only in M region of VMs, and not in Purkinje myocytes. TTX-R channels were found in M and ID, but their biophysical properties differed depending on recording location. Sodium current in rat VMs was upregulated by TNF-alpha. The magnitude of current increase was largest in M, but this difference was abolished by 100 nM TTX. CONCLUSIONS: Our data suggest that: a) a large fraction of TTX-R (likely Na(v)1.5) channels in the M region of VMs are inactivated at normal resting potential, leaving most of the burden of excitation to TTX-R channels in the ID; b) cell-cell adhesion increases functional channel density at ID. c) TTX-S (likely non-Na(v)1.5) channels make a minimal contribution to sodium current under control conditions, but represent a functional reserve that can be upregulated by exogenous factors

PURPOSE: We present a strategy to survey proteolytic processes that occur in human cancer microenvironments. EXPERIMENTAL DESIGN: In situ microdialysis during oral cancer surgery was combined with mass spectrometry-based proteomics to analyze interstitial fluid surrounding tumors and anatomically matched normal sites. Protease activity-based (18)O-profiling was utilized to reveal peptides that were processed by co-collected proteases ex vivo. RESULTS: We demonstrated for the first time the use of microdialysis in humans to collect interstitial fluid from cancer microenvironments. Proteomic profiling identified proteases and inhibitors in the microdialysis samples. A subset of peptides displayed characteristic (18)O-isotope patterns that indicated processing by endogenous proteases. CONCLUSIONS AND CLINICAL RELEVANCE: The presented approach provides unprecedented views of in vivo targets of proteases without disrupting the cancer or surrounding tissue. The methodology can be broadly adapted to other physiological conditions in which proteolytic mediators are involved (e.g. arthritic joints, inflamed muscle, other types of cancer) and where a comparison of normal and pathological tissue is sought.

Background: Reductions in brain-derived neurotrophic factor (BDNF) have been implicated in the pathophysiology of depression. Nevertheless, the factors influencing central and peripheral BDNF levels are still poorly understood. Cerebral microvascular endothelial cells are known to be a major source of BDNF within the brain. Exposure of these cells to amyloid beta (Abeta), which may play a role in the pathophysiology of late-life depression, results in cell death or injury with significant reductions in BDNF secretion. Moreover, in rodents, infusion of Abeta40 into the carotid artery resulted in a disruption of endothelial cells, which was not observed with Abeta42 infusion. Therefore, we hypothesized that concentrations of plasma Abeta40, but not Abeta42, would have a negative effect on plasma BDNF levels. Methods: We examined BDNF and Abeta levels in plasma via immunoblotting and ELISA assays, respectively, from 88 subjects with intact cognition (no dementia and a Mini-Mental State Exam score of at least 28) and no gross MRI abnormalities other than white matter hyperintensities. As these subjects were originally recruited for a study on major depressive disorder (MDD), 45 had MDD and 43 were age-matched controls. Results: Consistent with our prediction, Abeta40 levels were inversely correlated with BDNF concentrations (p<.001), whereas Abeta42 levels were independent of BDNF expression (p=.231). This pattern was similar when MDD and control subjects were analyzed separately. Discussion: Our results are consistent with the hypothesis that cerebral endothelial cells are a contributing source of peripheral BDNF and that their disruption by circulating Abeta40 results in reduction in BDNF. However, these preliminary findings need confirmation, and the mechanisms for our observation, including Abeta40-induced cerebral endothelial cell dysfunction, will have to be clarified

Color vision is found in many invertebrate and vertebrate species. It is the ability to discriminate objects based on the wavelength of emitted light independent of intensity. As it requires the comparison of at least two photoreceptor types with different spectral sensitivities, this process is often mediated by a mosaic made of several photoreceptor types. In this review, we summarize the current knowledge about the formation of retinal mosaics and the regulation of photopigment (opsin) expression in the fly, mouse, and human retina. Despite distinct evolutionary origins, as well as major differences in morphology and phototransduction machineries, there are significant similarities in the stepwise cell-fate decisions that lead from progenitor cells to terminally differentiated photoreceptors that express a particular opsin. Common themes include (i) the use of binary transcriptional switches that distinguish classes of photoreceptors, (ii) the use of gradients of signaling molecules for regional specializations, (iii) stochastic choices that pattern the retina, and (iv) the use of permissive factors with multiple roles in different photoreceptor types.

PURPOSE: Extended warm ischemia during partial nephrectomy can lead to considerable renal injury. Using a rat model of renal ischemia we examined the ability of a unique renoprotective cocktail to ameliorate warm ischemia-reperfusion injury. MATERIALS AND METHODS: A warm renal ischemia model was developed using 60 Sprague-Dawley(R) rats. The left renal artery was clamped for 40 minutes, followed by 48 hours of reperfusion. A renoprotective cocktail of a mixture of specific growth factors, mitochondria protecting biochemicals and Manganese-Porphyrin (MnTnHex-2-PyP(5+)) was given intramuscularly at -24, 0 and 24 hours after surgery. At 48 hours the 2 kidneys were harvested and examined with hematoxylin and eosin, and periodic acid-Schiff stains. Protein and gene expression were also analyzed to determine ischemia markers and the antioxidant response. RESULTS: Compared to ischemic controls, kidneys treated with the renoprotective cocktail showed significant reversal of morphological changes and a significant decrease in the specific ischemic markers lipocalin-2, mucin-1 and galectin-3. Quantitative reverse transcriptase-polymerase chain reaction revealed up-regulation of several antioxidant genes in treated animals. CONCLUSIONS: According to histopathological and several molecular measures our unique renoprotective cocktail mitigated ischemia-reperfusion injury

Acetylcholinesterase inhibitors are first-line therapies for Alzheimer's disease. These drugs increase cholinergic tone in the target areas of the cholinergic neurons of the basal forebrain. Basal forebrain cholinergic neurons are dependent upon trophic support by nerve growth factor (NGF) through its neurotrophin receptor, TrkA. In the present study, we investigated whether the acetylcholinesterase inhibitors donepezil and galantamine could influence neurotrophin receptor signaling in the brain. Acute administration of donepezil (3 mg/kg, i.p.) led to the rapid autophosphorylation of TrkA and TrkB neurotrophin receptors in the adult mouse hippocampus. Similarly, galantamine dose-dependently (3, 9 mg/kg, i.p.) increased TrkA and TrkB phosphorylation in the mouse hippocampus. Both treatments also increased the phosphorylation of transcription factor CREB and tended to increase the phosphorylation of AKT kinase but did not alter the activity of MAPK42/44. Chronic treatment with galantamine (3 mg/kg, i.p., 14 days), did not induce changes in hippocampal NGF and BDNF synthesis or protein levels. Our findings show that acetylcholinesterase inhibitors are capable of rapidly activating hippocampal neurotrophin signaling and thus suggest that therapies targeting Trk signaling may already be in clinical use in the treatment of AD

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling

Background- The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K(ATP)) channels in these myocytes have not been systemically investigated. Methods and Results- We recorded K(ATP) channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K(ATP) channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K(ATP) channel had a prolonged interburst closed time. CCS K(ATP) channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K(ATP) channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions- These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K(ATP) channel opening during periods of ischemia

The question of what modulates the firing of the cerebellar nuclei (CN) is one to which we presently have a surprisingly incomplete answer. Because most synaptic input to the CN originates from Purkinje cells (PCs), and simple spikes (SSs) are far more numerous than complex spikes (CSs), SSs are generally thought to be the dominant influence on the CN. However, evidence, reviewed here, suggests that this appears not to be the case in some physiologically important situations. As an alternative, we propose that CS activity may have at least as significant an effect on CN firing as do SSs. In particular, we suggest that CS activity has a role in controlling the bursts CN neurons show during various movements, during sleep states, and under ketamine-xylazine anesthesia. The ability to perform this role rests on the fact that CSs can be highly synchronized among PCs that project to the same CN neuron. Specifically, we suggest that synchronized CSs help determine the temporal course of the CN bursts, most often their offset, and that SSs and activity from cerebellar afferents may modulate the specific firing pattern within each burst. This joint control of CN activity may help explain anomalies present in the standard model for synaptic control of CN activity in which determination of CN firing patterns is attributed primarily to SSs