Spike frequency adaptation of rat hindlimb motoneurons.

The objective of our study was to resolve two issues pertaining to motoneuron (MN) spike frequency adaptation (SFA): 1) to develop an index of SFA that is sensitive to a wide range of adaptation patterns and would correlate well with MN excitability and 2) to determine whether SFA pattern is stimulus current dependent. Sprague-Dawley rats (250-350 g) were anesthetized (ketamine-xylazine) before electrophysiological properties from sciatic nerve MNs located in the lumbar spinal cord were recorded. SFA was measured by 30-s square-wave current injections at 1.5, 3.0, and 5.0 nA above estimated rhythmic firing threshold. Discharges per second were significantly (P < 0.001) higher for 5-nA than for 1.5- and 3-nA currents > rhythmic firing threshold in the first 2 s. SFA was quantified by using ratios of the final to initial number of discharges with 1-, 2-, and 5-s bins. The best index of SFA was the percent decline in the number of spikes fired in the fifth 5-s bin relative to the first 5-s bin [1 - (bin 5/bin 1)]. With the use of this index, we found that SFA was significantly correlated with several measures of MN excitability, including estimated persistent inward current amplitude (r = -0.76) and rheobase current (r = 0.71), and tended to correlate with input resistance (r = -0.43) and frequency-current slope (r = -0.57). This index also showed the widest range of SFA among MNs. In conclusion, an SFA pattern can be ascertained for each MN and becomes more pronounced as MN excitability decreases. Finally, for the first time, we report evidence of a relationship between persistent inward current and SFA.     

Water and nitrogen addition differentially impact plant competition in a native rough fescue grassland

We examined how water and nitrogen addition and water–nitrogen interactions affect root and shoot competition intensity and competition–productivity relationships in a native rough fescue grassland in central Alberta, Canada. Water and nitrogen were added in a factorial design to plots and root exclusion tubes and netting were used to isolate root and shoot competition on two focal species (Artemisia frigida and Chenopodium leptophyllum). Both water and nitrogen were limiting to plant growth, and focal plant survival rates increased with nitrogen but not water addition. Relative allocation to root biomass increased with water addition. Competition was almost entirely belowground, with focal plants larger when released from root but not shoot competition. There were no significant relationships between productivity and root, shoot, or total competition intensity, likely because in this system shoot biomass was too low to cause strong shoot competition and root biomass was above the levels at which root competition saturates. Water addition had few effects on the intensity of root competition suggesting that root competition intensity is invariant along soil moisture gradients. Contrary to general expectation, the strength of root competition increased with nitrogen addition demonstrating that the relationship between root competition intensity and nitrogen is more complex than a simple monotonic decline as nitrogen increases. Finally, there were few interactions between nitrogen and water affecting competition. Together these results indicate that the mechanisms of competition for water and nitrogen likely differ.     

Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip

Dilated cardiomyopathy (DCM) is a myocardial disease characterized by progressive depression of myocardial contractile function and ventricular dilatation. Thirty percent of DCM patients belong to the inherited genetic form; the rest may be idiopathic, viral, autoimmune, or immune-mediated associated with a viral infection. Disturbances in humoral and cellular immunity have been described in cases of myocarditis and DCM. A number of autoantibodies against cardiac cell proteins have been identified in DCM. In this study, we have profiled the autoantibody repertoire of plasma from DCM patients against a human protein array consisting of 37,200 redundant, recombinant human proteins and performed qualitative and quantitative validation of these putative autoantigens on protein microarrays to identify novel putative DCM specific autoantigens. In addition to analyzing the whole IgG autoantibody repertoire, we have also analyzed the IgG3 antibody repertoire in the plasma samples to study the characteristics of IgG3 subclass antibodies. By combining screening of a protein expression library with protein microarray technology, we have detected 26 proteins identified by the IgG antibody repertoire and 6 proteins bound by the IgG3 subclass. Several of these autoantibodies found in plasma of DCM patients, such as the autoantibody against the Kv channel-interacting protein, are associated with heart failure.     

A role for cyclic AMP in entrainment of the circadian oscillator in Xenopus retinal photoreceptors by dopamine but not by light

The circadian oscillator in Xenopus retinal photoreceptor layers can be reset in similar ways by light and agonists of D2-like dopamine receptors. Treatments that increase cyclic AMP levels act on this oscillator in an opposite fashion, mimicking darkness in the induction of phase shifts. Light and dopamine have each been reported to inhibit adenylate cyclase in photoreceptors. Together, these data suggest that the transduction pathways for entrainment by dopamine and/or light include suppression of cyclic AMP or a cyclic AMP-sensitive step. In these studies, we examined this hypothesis by measuring the effects of treatment with a cyclic AMP analogue on the phase shifts induced in photoreceptor melatonin rhythms by light or a D2 receptor agonist (quinpirole). When photoreceptor layers were treated simultaneously with 8-(4-chlorophenylthio)cyclic AMP (8-CPT-cAMP) and quinpirole at any of three different phases of the circadian cycle, the resulting phase shifts of the melatonin rhythm were always the same as those caused by 8-CPT-cAMP alone. This indicates that there is a cyclic AMP-sensitive step in the dopamine entrainment pathway. In contrast, light pulses did reset the oscillator in the presence of elevated cyclic AMP. This suggests a separate cyclic AMP-insensitive transduction pathway for entrainment by light. Quinpirole reduced basal levels of cyclic AMP in photoreceptors, but light did not. These data suggest that cyclic AMP plays a role in the entrainment pathway activated by dopamine but not in the entrainment pathway activated by light.     

Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations

Hypoxanthine (Hx) with specific (15)N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H (1)H and (15)N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the (1)H and (15)N chemical shifts of N-1H in the PNP.Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance approximately 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP.Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.     

An evaluation of detergents for NMR structural studies of membrane proteins

Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D (1)H-(15)N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested - R. sphaeroides LH1 alpha and beta subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter - with 1, 2, or 4 membrane spanning alpha-helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 beta subunits form native-like complexes with bacteriochlorophyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of (15)N transverse, longitudinal and (15)N[(1)H] nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15-20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed.