Feeling by sight or seeing by touch?

We have addressed the role of occipital and somatosensory cortex in a tactile discrimination task. Sight-ed and congenitally blind subjects rated the roughness and distance spacing for a series of raised dot patterns. When judging roughness, intermediate dot spacings were perceived as being the most rough, while distance judgments generated a linear relation. Low-frequency rTMS applied to somatosensory cortex disrupted roughness without affecting distance judgments, while rTMS to occipital cortex disrupted distance but not roughness judgments. We also tested an early blind patient with bilateral occipital cortex damage. Her performance on the roughness determination task was normal; however, she was greatly impaired with distance judgments. The findings suggest a double-dissociation effect in which roughness and distance are primarily processed in somatosensory and occipital cortex, respectively. The differential effect of rTMS on task performance and corroborative clinical evidence suggest that occipital cortex is engaged in tactile tasks requiring fine spatial discrimination.     

A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains.     

ApoE and clusterin cooperatively suppress Abeta levels and deposition: evidence that ApoE regulates extracellular Abeta metabolism in vivo

Apolipoprotein E (apoE) and clusterin can influence structure, toxicity, and accumulation of the amyloid-beta (Abeta) peptide in brain. Both molecules may also be involved in Abeta metabolism prior to its deposition. To assess this possibility, we compared PDAPP transgenic mice that develop age-dependent Abeta accumulation in the absence of apoE or clusterin as well as in the absence of both proteins. apoE(-/-) and clusterin(-/-) mice accumulated similar Abeta levels but much less fibrillar Abeta. In contrast, apoE(-/-)/clusterin(-/-) mice had both earlier onset and markedly increased Abeta and amyloid deposition. Both apoE(-/-) and apoE(-/-)/clusterin(-/-) mice had elevated CSF and brain interstitial fluid Abeta, as well as significant differences in the elimination half-life of interstitial fluid Abeta measured by in vivo microdialysis. These findings demonstrate additive effects of apoE and clusterin on influencing Abeta deposition and that apoE plays an important role in regulating extracellular CNS Abeta metabolism independent of Abeta synthesis.     

FcgammaRIIb-mediated negative regulation of BCR signalling is associated with the recruitment of the MAPkinase-phosphatase, Pac-1, and the 3'-inositol phosphatase, PTEN

The low-affinity receptor for IgG, FcgammaRIIb, negatively regulates B cell antigen receptor (BCR)-mediated proliferative signalling. FcgammaRIIb has been reported to mediate this inhibition by uncoupling the BCR from the RasMAPkinase pathway. We now show that FcgammaRIIb-mediated negative feedback inhibition also correlates with induction of an Erk-associated phosphatase activity that reflects the rapid association of Erk and the MAPkinase phosphatase, Pac-1, and dephosphorylation and inactivation of ErkMAPkinase. This mechanism of abrogating ongoing ErkMAPkinase signalling therefore provides a rationale for rapid immune-complex-mediated feedback inhibition of active antigen-driven B cell responses. In addition, FcgammaRIIb signalling also induces the recruitment and activation of the 3'-inositol phosphatase, PTEN, which by antagonising PI 3kinase activity and inhibiting BCR-coupling to the anti-apoptotic kinase, Akt, provides an additional mechanism for FcgammaRIIb-mediated negative regulation of BCR-coupling to ErkMAPkinase, cell survival and proliferation.     

Integrated data acquisition system for medical device testing and physiology research in compliance with good laboratory practices

In seeking approval from the US Food and Drug Administration (FDA) for clinical trial evaluation of an experimental medical device, a sponsor is required to submit experimental findings and support documentation to demonstrate device safety and efficacy that are in compliance with Good Laboratory Practices (GLP). The objective of this project was to develop an integrated data acquisition (DAQ) system and documentation strategy for monitoring and recording physiological data when testing medical devices in accordance with GLP guidelines mandated by the FDA. Data aquisition systems were developed as stand-alone instrumentation racks containing transducer amplifiers and signal processors, analog-to-digital converters for data storage, visual display and graphical user-interfaces, power conditioners, and test measurement devices. Engineering standard operating procedures (SOP) were developed to provide a written step-by-step process for calibrating, validating, and certifying each individual instrumentation unit and the integrated DAQ system. Engineering staff received GLP and SOP training and then completed the calibration, validation, and certification process for the individual instrumentation components and integrated DAQ system. Eight integrated DAQ systems have been successfully developed that were inspected by regulatory affairs consultants and determined to meet GLP guidelines. Two of these DAQ systems were used to support 40 of the pre-clinical animal studies evaluating the AbiCor artificial heart (ABIOMED, Danvers, MA). Based in part on these pre-clinical animal data, the AbioCor clinical trials began in July 2001. The process of developing integrated DAQ systems, SOP, and the validation and certification methods used to ensure GLP compliance are presented in this article.     

Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin

Two dimensional (2D) solid-state (13)C.(13)C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B(1) fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish (13)C-(13)C correlations in rhodopsin. In rhodopsin containing 4'-(13)C-Tyr and 8,19-(13)C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-(13)CH(3), which are 4.8 A apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-(13)CH(3), which are 5.5 A apart in the crystal structure. These data demonstrate that long range (13)C em leader (13)C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-(13)C Gly121 and U-(13)C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 A) in the crystal structure. Based on model compounds, the absence of a (13)C em leader (13)C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.     

Estimating the Location and Time Course of Synaptic Input from Multi-Site Potential Recordings

A method is introduced that permits accurate and robust extraction of the location and time course of synaptic conductance from potentials recorded on either side of, and perhaps at some distance from, the synapse in question. It is shown that such data permits one to fully overcome the problems typically associated with lack of spaceclamp. The method does not presume anything about the nature of the time course and yet is applicable to branched, active cells receiving simultaneous input from a number of synapses.     

Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response

The cell wall component lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis is involved in the inhibition of phagosome maturation, apoptosis and interferon (IFN)-gamma signalling in macrophages and interleukin (IL)-12 cytokine secretion of dendritic cells (DC). All these processes are important for the host to mount an efficient immune response. Conversely, LAM isolated from non-pathogenic mycobacteria (PILAM) have the opposite effect, by inducing a potent proinflammatory response in macrophages and DCs. LAMs from diverse mycobacterial species differ in the modification of their terminal arabinose residues. The strong proinflammatory response induced by PILAM correlates with the presence of phospho-myo-inositol on the terminal arabinose. Interestingly, recent work indicates that the biosynthetic precursor of LAM, lipomannan (LM), which is also present in the cell wall, displays strong proinflammatory effects, independently of which mycobacterial species it is isolated from. Results from in vitro assays and knock-out mice suggest that LM, like PILAM, mediates its biological activity via Toll-like receptor 2. We hypothesize that the LAM/LM ratio might be a crucial factor in determining the virulence of a mycobacterial species and the outcome of the infection. Recent progress in the identification of genes involved in the biosynthesis of LAM is discussed, in particular with respect to the fact that enzymes controlling the LAM/LM balance might represent targets for new antitubercular drugs. In addition, inactivation of these genes may lead to attenuated strains of M. tuberculosis for the development of new vaccine candidates.