Bioinformatics methods with subsequent verification by experimental data were applied to the structural investigation of the intracellular loop of the delta-subunit of the nicotinic acetylcholine receptor (nAChR). Three complementary methods were used: prediction of secondary structure elements, prediction of ordered/disordered protein regions and prediction of short functional binding motifs. The output of five different algorithms was used for the secondary structure construction. Most of the intracellular domain is predicted to be unfolded. The predictions correlate well with the experimental data of limited proteolysis and NMR performed on the mostly monomeric fraction of heterologously expressed Torpedo intracellular domain protein. Twelve functional binding motifs within the disordered regions of the nAChR intracellular domain are predicted. Identification of proteins that interact with the intracellular domain will provide a better understanding of protein-protein interactions involved in nAChR assembly, trafficking and clustering.

There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR). However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown. We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the delta-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs. To facilitate purification, (His)6-tags were attached with or without linkers, and the effects of protein truncations at C- or N-termini were examined. The proteins were purified from inclusion bodies under denaturing conditions by Ni-NTA-chromatography. Molecular weight and peptide mass fingerprint was determined by MALDI mass spectrometry. Size-exclusion chromatography revealed that the Torpedo intracellular delta-loop refolded in an aqueous buffer was present in solution as a dimer. Phosphorylation of this protein with protein kinase A and tyrosine kinase (Abl) occurred at the same serine and tyrosine residues as in the native receptor. According to CD spectra, the secondary structure was not sensitive to phosphorylation. The rat intracellular loops could be solubilized only in the presence of non-ionic detergents or lipids. CD spectra indicate that the Torpedo and rat proteins have differences in their secondary structure. In the presence of dodecylphosphocholine, high concentrations (up to 6 mg/ml) of the Torpedo and rat intracellular loops were achieved. The results suggest that the spatial structure of the intracellular loops is dependent on environment and species, but is not changed significantly upon enzymatic phosphorylation.

title influence of chromium on rat brain 5-hydroxytryptamine R. Venkatakrishna Murali Dr ALM PGIBMS, University of Madras, Taramani, Chennai, India While health effects of chromium like allergic dermatitis, sensitization reaction of the skin, chrome ulcers, carcinogenesis of lungs are known, reports regarding its effect on central nervous system are scanty. In the present study, the impact of hexavalent chromium on CNS was assessed by its effect on 5-HT (serotonin), since tryptaminergic neurons which diffusely innervate most regions of the CNS synthesize, store, and release 5-HT as a transmitter. Separate groups of Wistar albino rats were treated with potassium dichromate daily for 15, 45, and 90 days by oral route and one group was allowed a recovery period of 60 days following 90 days of treatment with chromium. 5-HT and its metabolite 5-hydroxyindoleaceticacid (5-HIAA) in brain tissue were analysed. Chromium treatment for 15 days resulted in moderate increase in brain 5-HT and 5-HIAA indicating an enhanced turnover of 5-HT in brain. When exposure duration was extended to 45 days, there was a decrease in 5-HT accompanied by an increase in 5-HIAA indicating an augmented breakdown of 5-HT. Prolonged daily exposure to chromium for 90 days decreased the turnover of 5-HT since there was a reduction in 5-HT as well as 5-HIAA. Thus the nature of chromium influence on brain 5-HT is found to depend on duration of exposure. The effect of chromium seems to persist even after exposure since there was a significant reduction in the 5-HIAA even after the recovery period. The clinical implications of these observations need consideration since brain tryptaminergic neurons are involved in various functions such as mood, sleep, appetite, etc. and 5-HT is also involved in conditions such as depression, anxiety, and migraine.

On the structural map of the nicotinic acetylcholine receptor (nAChR) the intracellular domain remains "a blank space". In order to investigate the structure and function of this intracellular domain we are expressing the intracellular loop of the nAChR delta-subunit individually and the loops of all subunits (alpha(2)betagammadelta) collectively, using a polycistronical vector. E.coli cells express the individual loop in insoluble form, after refolding and purification a protein concentration of 0.3 mg/ml can be obtained. The CD spectra of the fusion protein show a high percentage of ordered structure. The coexpression of the loops in the same cell did not improve protein solubility, after refolding the protein yield remains low (similar to0.05 mg/ml). By cross-linking experiments the formation of distinct oligomers has been demonstrated.

Exploratory measurements have been made of the intensity of several forbidden neutron reflections simulated by multiple Bragg reflection in large single crystals. The (0003) and (0001) reflections were studied in a beryllium crystal with mosaic 0.25\ifmmode^\circ\else\textdegree\fi{} and the (200) in a germanium crystal with mosaic 0.02\ifmmode^\circ\else\textdegree\fi{}. Double-crystal methods were used with a triple-axis spectrometer. Large amounts of order contamination were treated by means of resonance absorption filters and least-squares analysis of overdetermined equations. To minimize the effect of peculiarities of individual specimens, the results are presented as intensity ratios. Simulated reflections were observed to have 1/60 to $\frac{1}{5}$ the intensity of ordinary Bragg reflections. No conclusive evidence was found for an intrinsic component due to the forbidden reflections themselves; the Be simulations were at least 200 to 700 times, and the Ge simulations at least 15 to 60 times, more intense than the corresponding intrinsic components. Caution must be used in applying these results to other specimens.