Differences in monoamine oxidase activity between cultured noradrenergic and cholinergic sympathetic neurons

Two types of monoamine oxidase activity (MAO-A and MAO-B) help regulate the levels of biogenic amines such as catecholamines and serotonin. Although MAO-A has greater activity toward most catecholamines than MAO-B, no direct experiments have determined the types and levels of MAO activity that are normally expressed in noradrenergic neurons. Noradrenergic neurons from neonatal rat superior cervical ganglia were isolated and cultured under conditions that permit either continued expression of the noradrenergic phenotype or promote a transition to a predominantly cholinergic phenotype. After 14-21 days in vitro, neurons from both types of cultures were assayed for the type and amount of monoamine oxidase activity using tryptamine, a common substrate for both MAO-A and MAO-B. Neurons cultured under noradrenergic conditions expressed sevenfold greater MAO activity than neurons cultured under cholinergic conditions. Essentially all MAO activity in the noradrenergic cultures was inhibited by preincubation with 10(-8)-10(-9) M clorgyline, which indicated that this activity was primarily MAO-A. Cultures grown under cholinergic conditions exhibited 6- to 10-fold lower MAO-A activity and an 8- to 10-fold lower level of catecholamine synthesis from labeled precursors compared to neurons grown under noradrenergic conditions. These results directly demonstrate that high MAO-A activity is expressed in noradrenergic neurons in vitro. The corresponding decreases in both MAO-A specific activity and catecholamine synthesis as neurons become cholinergic in vitro suggest that the expression of the noradrenergic phenotype involves the coordinate regulation of degradative as well as synthetic enzymes involved in catecholamine metabolism.     

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure,backbone dynamics and protein association

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155–270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of¹H-¹⁵N-correlated 2D HSQC,¹⁵N-edited 3D TOCSY-HMQC, and¹⁵N-edited 3D NOESY-HMQC spectra. By analysis of the -proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain.To investigate the internal dynamics of the protein backbone, T₁ and T₂ relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.Abbreviations SH Src homology - GST glutathione-S-transferase - IPTG isopropyl--D-galactopyranoside - DTT dithiothreitol - PMSF phenyl-methyl-sulphonyl-fluoride - TBS 50 mM Tris, 150 mM NaCl, 5 mM DTT, pH 8.0 - MWCO molecular weight cut off - NMR nuclear magnetic resonance - HSQC heteronuclear single-quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Nuclear magnetic resonance analysis of water in natural and deuterated mouse muscle above and below freezing.

Measurements of absolute proton signal intensities, free induction decays, spin-spin relaxation times, and local fields in the rotating frame in natural and fully deuterated mouse muscle at five temperatures in the range 293-170 K are reported. The analysis is carried out at three time windows on the free induction decay. The contribution to the magnetization from protons on large molecules and water are analyzed.     

Spectroscopic and binding studies of azide to type-2-copper-depleted ascorbate oxidase from zucchini

Summary Binding of azide to type-2-copper-depleted (T2D) zucchini ascorbate oxidase, containing reduced type-3 Cu centers, and met-T2D ascorbate oxidase, containing oxidized type-3 Cu centers, has been studied spectroscopically. In both cases titration with azide in 0.1 M phosphate pH 6.8 produces a broad near-ultraviolet band with maximum at 455 nm (e 2500 M^–1 cm^–1, with respect to the met-T2D enzyme) and shoulder at 390 nm (e 1700 M^–1 cm^–1), that are assigned to(azide)Cu(II) ligand-to-metal charge transfer (LMCT) transitions. This is accompanied by a reduction of absorbance at 330 nm in the met-T2D) enzyme adduct (e –1400 M^–1 cm^–1). A broad circular dichroic band of negative sign between 370–480 nm corresponds to the LMCT absorption band. Analysis of the titration data indicates that one azide ion binds independently to each of the binuclear T3 Cu couples with low affinity (K = 50 M^–1). The ESR signal of the T1 Cu observed in frozen solutions of the T2D enzyme is also perturbed by the addition of azide. The analogies in the azide-binding characteristics between ascorbate oxidase and laccase are discussed.     

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides

Cellular and Molecular Neurobiology - Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses....     

Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice

Gene targeting was used to delete exon 2 of mouse DOR-1, which encodes the delta opioid receptor. Essentially all 3H-[D-Pen2,D-Pen5]enkephalin (3H-DPDPE) and 3H-[D-Ala2,D-Glu4]deltorphin (3H-deltorphin-2) binding is absent from mutant mice, demonstrating that DOR-1 encodes both delta1 and delta2 receptor subtypes. Homozygous mutant mice display markedly reduced spinal delta analgesia, but peptide delta agonists retain supraspinal analgesic potency that is only partially antagonized by naltrindole. Retained DPDPE analgesia is also demonstrated upon formalin testing, while the nonpeptide delta agonist BW373U69 exhibits enhanced activity in DOR-1 mutant mice. Together, these findings suggest the existence of a second delta-like analgesic system. Finally, DOR-1 mutant mice do not develop analgesic tolerance to morphine, genetically demonstrating a central role for DOR-1 in this process.     

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH₂, where X = arginine, N^ω,N^ω-dimethylarginine, DMA, or lysine) corresponding to residues 676–692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N^ω,N^ω-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.     

Geometric and mechanical properties of human cervical spine ligaments

This study characterized the geometry and mechanical properties of the cervical ligaments from C2-T1 levels. The lengths and cross-sectional areas of the anterior longitudinal ligament, posterior longitudinal ligament, joint capsules, ligamentum flavum, and interspinous ligament were determined from eight human cadavers using cryomicrotomy images. The geometry was defined based on spinal anatomy and its potential use in complex mathematical models. The biomechanical force-deflection, stiffness, energy, stress, and strain data were obtained from 25 cadavers using in situ axial tensile tests. Data were grouped into middle (C2-C5) and lower (C5-T1) cervical levels. Both the geometric length and area of cross section, and the biomechanical properties including the stiffness, stress, strain, energy, and Young's modulus, were presented for each of the five ligaments. In both groups, joint capsules and ligamentum flavum exhibited the highest cross-sectional area (p < 0.005), while the longitudinal ligaments had the highest length measurements. Although not reaching statistical significance, for all ligaments, cross-sectional areas were higher in the C5-T1 than in the C2-C5 group; and lengths were higher in the C2-C5 than in the C5-T1 group with the exception of the flavum (Table 1 in the main text). Force-deflection characteristics (plots) are provided for all ligaments in both groups. Failure strains were higher for the ligaments of the posterior (interspinous ligament, joint capsules, and ligamentum flavum) than the anterior complex (anterior and posterior longitudinal ligaments) in both groups. In contrast, the failure stress and Young's modulus were higher for the anterior and posterior longitudinal ligaments compared to the ligaments of the posterior complex in the two groups. However, similar tendencies in the structural responses (stiffness, energy) were not found in both groups. Researchers attempting to incorporate these data into stress-analysis models can choose the specific parameter(s) based on the complexity of the model used to study the biomechanical behavior of the human cervical spine.     

Moment-rotation responses of the human lumbosacral spinal column

The objective of this study was to test the hypothesis that the human lumbosacral joint behaves differently from L1-L5 joints and provides primary moment-rotation responses under pure moment flexion and extension and left and right lateral bending on a level-by-level basis. In addition, range of motion (ROM) and stiffness data were extracted from the moment-rotation responses. Ten T12-S1 column specimens with ages ranging from 27 to 68 years (mean: 50.6+/-13.2) were tested at a load level of 4.0 N m. Nonlinear flexion and extension and left and right lateral bending moment-rotation responses at each spinal level are reported in the form of a logarithmic function. The mean ROM was the greatest at the L5-S1 level under flexion (7.37+/-3.69 degrees) and extension (4.62+/-2.56 degrees) and at the L3-L4 level under lateral bending (4.04+/-1.11 degrees). The mean ROM was the least at the L1-L2 level under flexion (2.42+/-0.90 degrees), L2-L3 level under extension (1.58+/-0.63 degrees), and L1-L2 level under lateral bending (2.50+/-0.75 degrees). The present study proved the hypothesis that L5-S1 motions are significantly greater than L1-L5 motions under flexion and extension loadings, but the hypothesis was found to be untrue under the lateral bending mode. These experimental data are useful in the improved validation of FE models, which will increase the confidence of stress analysis and other modeling applications.