Maintenance of high-frequency transmission at purkinje to cerebellar nuclear synapses by spillover from boutons with multiple release sites

Cerebellar Purkinje neurons maintain high firing rates but their synaptic terminals depress only moderately, raising the question of how vesicle depletion is minimized. To identify mechanisms that limit synaptic depression, we evoked 100 Hz trains of GABAergic inhibitory postsynaptic currents (IPSCs) in cerebellar nuclear neurons by stimulating Purkinje axons in mouse brain slices. The paired-pulse ratio (IPSC(2)/IPSC(1)) of the total IPSC was approximately 1 and the steady-state ratio (IPSC(20)/IPSC(1)) was approximately 0.5, suggesting a high response probability of postsynaptic receptors, without an unusually high release probability. Three-dimensional electron microscopic reconstructions of Purkinje boutons revealed multiple active zones without intervening transporters, suggestive of "spillover"-mediated transmission. Simulations of boutons with 10-16 release sites, in which transmitter from any site can reach all receptors opposite the bouton, replicated multiple-pulse depression during normal, high, and low presynaptic Ca influx. These results suggest that release from multiple-site boutons limits depletion-based depression, permitting prolonged, high-frequency inhibition at corticonuclear synapses.     

sGAL: a computational method for finding surface exposed sites in proteins suitable for Cys-mediated cross-linking

sGAL is a computer program designed to find pairs of sites suitable for introducing chemical cross-links into proteins. sGAL takes a protein structure file in PDB format as input, truncates each residue sequentially to its gamma side chain atom to mimic mutation to Cys, and calculates the exposed surface area of the gamma atom. The user then inputs the minimum and maximum lengths of the cross-linker. sGAL provides as output pairs of residues that would have exposed gamma atom separations that fall within this range. Furthermore, if a line joining the pair of gamma atoms contacts more than a given number of buried atoms, that pair is discarded. In this way, sites for which the protein would sterically interfere with cross-linking are avoided. AVAILABILITY: http://www.chem.utoronto.ca/staff/GAW/links.html; (Surface Racer is also required see: http://monte.biochem.wisc.edu/~tsodikov/surface.html).     

II. Novel deaminated sulfadiazine metabolites in neonatal calf tissue,plasma, and urine following oral treatment with 14C-sulfadiazine

Demonstration of the efficacy of Tribrissen® [trimethoprim(TMP): sulfadiazine(SDZ), 1:5] for treatment of calf scours led to a study of the disposition of ¹⁴C-SDZ in the neonatal calf. Two healthy male calves (～40 kg) received orally one Tribrissen® bolus [¹⁴C-SDZ (1.0 g):TMP (0.2 g)] each for five consecutive days. The calves were killed on day 14 after the last dose. Mean total SDZ-related residues were 0.04 ppm (muscle), 0.16 ppm (kidney) and 0.31 ppm (liver) on day 14. Intact SDZ was undetectable on day 14 in all three tissues. Two-dimensional TLC autoradiograms of tissue, plasma and urine extracts revealed the presence of SDZ, N⁴-acetyl SDZ, 4-hydroxy-SDZ, N⁴-acetyl-4-hydroxy SDZ and two novel metabolites that were identified by NMR and mass spectral analyses to be 2-benzenesulfonamidopyrimidine and 2-benzenesulfonamido-4-hydroxypyrimidine. Most of the SDZ dose (87%) was recovered in the urine with SDZ and N⁴-acetyl SDZ accounting for 16 and 42% of the dose. The other four compounds accounted for <5% of the dose. The data from this study indicate that assays that rely on the presence of the N⁴-amino group, such as the Bratton-Marshall procedure, measure only part of the SDZ-related tissue residues in neonatal calves.     

The role of the extracellular loops of the CGRP receptor, a family B GPCR

The CGRP (calcitonin gene-related peptide) receptor is a family B GPCR (G-protein-coupled receptor). It consists of a GPCR, CLR (calcitonin receptor-like receptor) and an accessory protein, RAMP1 (receptor activity-modifying protein 1). RAMP1 is needed for CGRP binding and also cell-surface expression of CLR. There have been few systematic studies of the ECLs (extracellular loops) of family B GPCRs. However, they are likely to be especially important for the interaction of the N-termini of the peptide agonists that are the natural agonists for these receptors. We have carried out alanine scans on all three ECLs of CLR, as well as their associated juxtamembrane regions. Residues within all three loops influence CGRP binding and receptor activation. Mutation of Ala203 and Ala206 on ECL1 to leucine increased the affinity of CGRP. Residues at the top of TM (transmembrane) helices 2 and 3 influenced CGRP binding and receptor activation. L351A and E357A in TM6/ECL3 reduced receptor expression and may be needed for CLR association with RAMP1. ECL2 seems especially important for CLR function; of the 16 residues so far examined in this loop, eight residues reduce the potency of CGRP at stimulating cAMP production when mutated to alanine.     

Reiteration in the Monodominant Tropical Tree Dicymbe corymbosa (Caesalpiniaceae) and its Potential Adaptive Significance

The tropical monodominant tree Dicymbe corymbosa reiterates via epicormic shoots and roots, resulting in multistemmed trees with complex pseudotrunks and root mounds. In 2 ha of primary forest on the Guiana Shield, we quantified the reiterative structure and aboveground soil development of 307 D. corymbosa individuals ≥ 10 cm dbh and investigated the potential adaptive significance of reiteration in terms of genet persistence and root exploitation of aboveground soil accumulations. We also investigated the incidence of the heart rot fungus Phellinus robustus in D. corymbosa and examined its relationship to the reiteration process. Large trees contained more and larger reiterations, greater trunk, root mound, and organic soil volumes, and a higher incidence of Phellinus than smaller trees. Roots and ectomycorrhizas were abundant in aboveground soils on the trees, occurred at higher densities than those of the surrounding forest floor, and may be important in recycling mineral nutrients. Stem turnover and reiteration were associated with Phellinus heart rot and appeared to be cumulative over time, resulting in persistent, structurally complex trees of indeterminate lifespan. Dicymbe corymbosa provides a rare example of a tree species that exploits both persistence and recruitment niches, as it successfully recruits through mast fruiting.     

Biosynthesis of ditigloyl esters of DI- and tri-hydroxytropanes in Datura

3α-Tigloyloxytropane-[¹⁴CO] [N-¹⁴Me], ratio 1·6:1 and valtropine-[¹⁴CO] [N-¹⁴Me], ratio 1·75:1 were separately fed via cotton wicks to 4-month-old Datura innoxia plants. After 8 days the root alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and the distribution of radioactivity in the acid and alkamine moieties was determined by hydrolysis. The precursor ratios were not maintained in the isolated ditigloyl esters, a result which does not support our hypothesis that the ditigloyl esters are formed by the progressive hydroxylation of 3α-tigloyloxytropane.     

The isolation and identification of a secreted biomarker associated with cell stress in serum‐free CHO cell culture

One approach to improving mammalian culture productivity has been to reduce cell stress and cell death in the bioreactor, thus enhancing productivity through a longer phase of viability. Here we describe the isolation and identification of a biomarker for stress and viability loss in CHO culture. Using SELDI‐TOF mass spectrometry to profile the protein component of supernatant culture media we have identified a peak at 7.7 kDa that was associated with loss of viability toward the end of the culture and simulated stress from both toxic metabolite accumulation and nutrient depletion. The relative intensity (signal/noise ratio) of the peak increased rapidly at the onset of dropping viability toward the end of the growth phase. Also, the peak height was seen to increase significantly when cells were grown under conditions emulating ammonia accumulation and glutamine deprivation. The species has been identified as a fragment of Galectin‐1 (Gal‐1) via MS/MS fingerprinting. We propose that this peak could be utilized as a marker for early onset of stress in cell culture. This work demonstrates the efficacy of SELDI technology to identify biomarkers in mammalian cell culture and highlights its value as a tool for the monitoring and improvement of culture processes. Biotechnol. Bioeng. 2009; 104: 590–600 © 2009 Wiley Periodicals, Inc.