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Systematic Mapping (systematic + mapping)

Distribution by Scientific Domains

Life Sciences	75%

Selected Abstracts

Characterization in vivo of bilaterally branching pontocerebellar mossy fibre to Golgi cell inputs in the rat cerebellum

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2009
Tahl Holtzman
Abstract Golgi cells regulate the flow of information from mossy fibres to the cerebellar cortex, through a mix of feedback and feedforward inhibitory actions on granule cells. The aim of the current study was to examine mossy fibre input to Golgi cells, in order to assess their impact on switching Golgi cells into feedforward behaviour. In urethane-anaesthetized rats, extracellular recordings were made from Golgi cells in Crus II (n = 18). Spikes were evoked in all Golgi cells by microstimulation within the contralateral hemispheral cortex, via branches of mossy fibres that terminate in both cerebellar hemispheres. The latencies of these responses were very short, consistent with a monosynaptic mossy fibre contact [average onset latency 2.3 ± 0.1 ms (SEM)]. The same stimuli had no measurable effect on spike responses of nearby Purkinje cells (n = 12). Systematic mapping in the contralateral cerebellar hemisphere (Crus Ib, IIa, IIb and the paramedian lobule) usually revealed one low-intensity stimulus ,hotspot' (12,35 ,A) from which short-latency spikes could be evoked in an individual Golgi cell. Microinjections of red and green retrograde tracers (latex beads, ,50,150 nL injection volume) made at the recording site and the stimulation hotspot resulted in double-labelled neurons within the pontine nuclei. Overall, this suggests that subsets of pontine neurons supply mossy fibres that branch to both hemispheres, some of which directly target Golgi cells. Such an arrangement may provide a common feedforward inhibitory link to temporally couple activity on both sides of the cerebellum during behaviour. [source]

Localization of nAChR subunit mRNAs in the brain of Macaca mulatta

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2000
Zhi-Yan Han
Abstract We present here a systematic mapping of nAChR subunit mRNAs in Macaca mulatta brain. A fragment, from the transmembrane segments MIII to MIV of Macaca neuronal nAChR subunits was cloned, and shown to exhibit high identity (around 95%) to the corresponding human subunits. Then, specific oligodeoxynucleotides were synthesized for in situ hybridization experiments. Both ,4 and ,2 mRNA signals were widely distributed in the brain, being stronger in the thalamus and in the dopaminergic cells of the mesencephalon. Most brain nuclei displayed both ,4 and ,2 signals with the exception of some basal ganglia regions and the reticular thalamic nucleus which were devoid of ,4 signal. ,6 and ,3 mRNA signals were selectively concentrated in the substantia nigra and the medial habenula. The strongest signals for ,3 or ,4 mRNAs were found in the epithalamus (medial habenula and pineal gland), whereas there were no specific ,3 or ,4 signals in mesencephalic dopaminergic nuclei. ,5 and ,7 mRNA signals were found in several brain areas, including cerebral cortex, thalamus and substantia nigra, although at a lower level than ,4 and ,2. The distribution of ,3, ,4, ,5, ,6, ,7, ,2, ,3 and ,4 subunit mRNAs in the monkey is substantially similar to that observed in rodent brain. Surprisingly, ,2 mRNA signal was largely distributed in the Macaca brain, at levels comparable with those of ,4 and ,2. This observation represents the main difference between rodent and Macaca subunit mRNA distribution and suggests that, besides ,4,2*, ,2,2* nAChRs constitute a main nAChR isoform in primate brain. [source]

Crystallization of IgG1 by mapping its liquid,liquid phase separation curves

BIOTECHNOLOGY & BIOENGINEERING, Issue 5 2006
Adam Idu Jion
Abstract Monoclonal antibody therapeutics is an important and fast expanding market. While production of these molecules has been a major area of research, much less is known regarding the stabilization of these proteins for delivery as drugs. Crystallization of antibodies is one such promising route for protein stabilization at high titers, and here we took a systematic approach to initiate crystallization through nucleation in a simple PEG (polyethylene glycol), protein in water solution. A ternary mixture of globular proteins, PEG, and water will undergo a liquid,liquid phase separation (LLPS) as shown in a phase diagram or a Binodal curve. Of particular interest within the phase diagram is the position of the critical point, which is where nucleation occurs most rapidly. Detailed LLPS maps were created by increasing concentrations of PEG (from 5% to 11%) and IgG (from 1 to 20 mg/mL). By increasing the molecular weight (MW) of PEG (and hence its radius of gyration) from 1,000 to 6,000 g/mol, the temperatures of the critical point of nucleation were shown to increase. Once these curves were determined, nucleation experiments were conducted close to a chosen critical point (10.5 mg/mL IgG in 11% PEG 1000) and after 3 weeks, crystals of IgG of approximately 100 ,m in size were successfully formed. This is the first example of crystallization of an antibody through systematic mapping of LLPS curves, which is a fundamental step towards the scale-up of antibody crystallization. © 2006 Wiley Periodicals, Inc. [source]

Mapping the Limits of Substrate Specificity of the Adenylation Domain of TycA

CHEMBIOCHEM, Issue 4 2009
Benoit R. M. Villiers
Abstract The limits and potential of substrate promiscuity of the adenylation domain of tyrocidine synthetase 1 were systematically explored. Substrate acceptance is governed by hydrophobic effects (as shown by the correlation of kcat/KM and side-chain log,P), shape complementarity and steric exclusion. The quantification of these factors provides ground rules for understanding and possibly evolving substrate specificity in this class of enzymes. The catalytic potential of tyrocidine synthetase 1 (TycA) was probed by the kinetic characterization of its adenylation activity. We observed reactions with 30 substrates, thus suggesting some substrate promiscuity. However, although the TycA adenylation (A) domain was able to accommodate alternative substrates, their kcat/KM values ranged over six orders of magnitude. A comparison of the activities allowed the systematic mapping of the substrate specificity determinants of the TycA A-domain. Hydrophobicity plays a major role in the recognition of substrate analogues but can be combined with shape complementarity, conferring higher activity, and/or steric exclusion, leading to substantial discrimination against larger substrates. A comparison of the kcat/KM values of the TycA A-domain and phenylalanyl-tRNA synthetase showed that the level of discrimination was comparable in the two enzymes for the adenylation reaction and suggested that TycA was also subjected to high selective pressure. The specificity patterns observed and the quantification of alternative activities provide a basis for exploring possible paths for the future directed evolution of A-domain specificity. [source]