Notes: Fluorene–Arn complexes formed in a pulsed supersonic jet have been studied in their S1 state using two color REMPI spectroscopy with mass resolved detection. The appearance and shifts of the S1 origins relative to the fluorene monomer are measured for cluster sizes up to n=30. The shifts and appearance of these bands are used to identify multiple conformations at low n and have indicated a shift from two sided clustering by Ar at low n to primarily one sided clustering at large n. The ionic ground state of the smaller clusters (n≤6) are studied using mass analyzed threshold ionization (MATI) spectroscopy. The change of the ionization potentials as a function of cluster size has been determined. In the case of the fluorene–Ar4 cluster, the MATI spectrum of two separate cluster conformations was measured, revealing significantly different ionization potentials. Vibrational dynamics has been studied in several smaller clusters (n≤3) by measuring MATI and ZEKE spectra when pumping vibronic transitions in the fluorene chromophore. Significantly enhanced coupling of the chromophore to van der Waals modes is observed in going from n=1 to n=3. © 1997 American Institute of Physics.

Notes: Using immunohistochemistry and in situ hybridization, we studied changes in expression of some neuropeptides in large and medium-sized neurons in lumbar 4 and 5 rat dorsal root ganglia projecting to the gracile nucleus, in response to peripheral axotomy. Fourteen days after unilateral sciatic nerve transection, many large neurons and some medium-sized neurons in ipsilateral dorsal root ganglia were strongly neuropeptide Y-positive. Galanin-, vasoactive intestinal polypeptide (VIP)- and peptide histidine-isoleucine (PHI)-like immunoreactivities coexisted with neuropeptide Y-like immunoreactivity in some of these neurons. After axotomy numerous large and medium-sized cells contained neuropeptide Y mRNA in the ipsilateral ganglia, whereas no hybridization was seen in the contralateral or control ganglia. Cross-sectioned, large neuropeptide Y-positive fibres were observed in a somatotopically appropriate zone within the ipsilateral gracile fasciculus. A dense network of neuropeptide Y-immunoreactive, large nerve fibres and terminals was seen in the ipsilateral gracile nucleus. A small number of galanin- and VIP/PHI-like immunoreactive nerve fibres and terminals were also observed in adjacent sections. Neuropeptide Y-like immunoreactivity colocalized with galanin- or VIP/PHI-like immunoreactivity in some nerve fibres. None of these neuropeptide immunoreactivities could be detected in nerve fibres and terminals in the control or contralateral gracile nucleus. These findings suggest that neuropeptides, in addition to their role in small dorsal root ganglion neurons, may have a function in large and medium-sized dorsal root ganglion neurons projecting to laminae III and IV in the dorsal horn as well as to the gracile nuclei, as a part of their response to peripheral axotomy.

Notes: Peripheral tissue injury-induced central sensitization may result from the altered biochemical properties of spinal dorsal horn. However, peripheral nerve injury-induced modification of genes in the dorsal horn remains largely unknown. Here we identified strong changes of 14 channels, 25 receptors and 42 signal transduction related molecules in Sprague–Dawley rat dorsal spinal cord 14 days after peripheral axotomy by cDNA microarray. Twenty-nine genes were further confirmed by semiquantitative RT-PCR, Northern blotting, in situ hybridization and immunohistochemistry. These regulated genes included Ca2+ channel α1E and α2/δ1 subunits, α subunits for Na+ channel 1 and 6, Na+ channel β subunit, AMAP receptor GluR3 and 4, GABAA receptor α5 subunit, nicotinic acetylcholine receptor α5 and β2 subunits, PKC α, βI and δ isozymes, JNK1–3, ERK2–3, p38 MAPK and BatK and Lyn tyrosine-protein kinases, indicating that several signal transduction pathways were activated in dorsal spinal cord by peripheral nerve injury. These results demonstrate that peripheral nerve injury causes phenotypic changes in spinal dorsal horn. Increases in Ca2+ channel α2/δ1 subunit, GABAA receptor α5 subunit, Na+ channels and nicotinic acetylcholine receptors in both dorsal spinal cord and dorsal root ganglia indicate their potential roles in neuropathic pain control.

Notes: Peripheral axotomy-induced sprouting of thick myelinated afferents (A-fibers) from laminae III–IV into laminae I–II of the spinal cord is a well-established hypothesis for the structural basis of neuropathic pain. However, we show here that the cholera toxin B subunit (CTB), a neuronal tracer used to demonstrate the sprouting of A-fibers in several earlier studies, also labels unmyelinated afferents (C-fibers) in lamina II and thin myelinated afferents in lamina I, when applied after peripheral nerve transection. The lamina II afferents also contained vasoactive intestinal polypeptide and galanin, two neuropeptides mainly expressed in small dorsal root ganglion (DRG) neurons and C-fibers. In an attempt to label large DRG neurons and A-fibers selectively, CTB was applied four days before axotomy (pre-injury-labelling), and sprouting was monitored after axotomy. We found that only a small number of A-fibers sprouted into inner lamina II, a region normally innervated by C-fibers, but not into outer lamina II or lamina I. Such sprouts made synaptic contact with dendrites in inner lamina II. Neuropeptide Y (NPY) was found in these sprouts in inner lamina II, an area very rich in Y1 receptor-positive processes. These results suggest that axotomy-induced sprouting from deeper to superficial layers is much less pronounced than previously assumed, in fact it is only marginal. This limited reorganization involves large NPY immunoreactive DRG neurons sprouting into the Y1 receptor-rich inner lamina II. Even if quantitatively small, it cannot be excluded that this represents a functional circuitry involved in neuropathic pain.

Notes: Using indirect immunofluorescence, neuropeptide Y Y1 receptor (Y1 receptor)-like immunoreactivity (LI) was localized close to the plasmalemma of small neurons in lumbar dorsal root ganglia (DRGs) and neurons in the inner lamina II of the lumbar spinal cord of the rat. Using confocal microscopy, colocalization of Y1 receptor-LI and transferrin receptor-LI, a marker for endosomes and coated vesicles, was observed in dot-like structures along the plasmalemma. Under the electron microscope, Y1 receptor-LI was localized in coated vesicles and endosomes, in the membrane of tubular cisternae, sometimes connected to multivesicular bodies, and in the plasmalemma. These complex distribution patterns may reflect receptor turnover and internalization processes. In the lamina II of the spinal dorsal horn, Y1 receptor-LI was localized in the plasmalemma of neurons without any apparent association with paramembrane structures, as described above for the DRG neurons. Many dendrites were Y1 receptor-positive, and some of them made synaptic contacts with unstained axonal terminals. In general, Y1 receptor-LI was localized in the membrane outside the postsynaptic density. Double-immunofluorescence staining showed that most Y1 receptor-immunoreactive neurons in lamina II contained somatostatin-LI. Both in DRG and dorsal horn neurons, the Y1 receptor thus seems to represent a postjunctional/postsynaptic receptor.

Notes: Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [125I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [125I]NPY binding was most strongly attenuated by NPY13–36, a Y2 agonist, and partially inhibited by [Leu31,Pro34]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY13–36 completely abolished PYY binding, which was also reduced by [Leu,31 Pro34] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu31, Pro34] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).

Notes: [Auszug] We used illegitimate transcription in lymphoblastoid cell lines6 to obtain laminin α2 chain mRNA from probands of consanguineous laminin α2 and non-consanguineous chain-deficient families for mutation analysis by RT-PCR. Twenty-four pairs of overlapping primers (see Methods) were ...

Notes: [Auszug] DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product ...