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The initial goal of this work was to verify the geometry of the product of a photochemical reaction, viz. the title compound, C(8)H(10)O(6)S, (II). Our crystallographic study firmly establishes the cis-anti-cis nature of the substituents on the cyclobutane ring. The geometry is also designated as exo, where exo signifies that the five-membered ring is on the opposite side of the central cyclobutane ring from the carboxylic acid substituents. The structure determination reveals two molecules, A and B, in the asymmetric unit that display substantially different conformations of the bicyclic core: the cyclobutane ring puckering angles are 22 and 3 degrees , and the sulfolane ring conformations are twist (S-exo) and envelope (S-endo). Intrigued by this variation, we then compared the conformations of other molecules in the Cambridge Structural Database that have sulfolane rings fused to cyclobutane rings. In this class of compound, there are five examples of saturated cyclobutane rings, with ring puckering angles ranging from 3 to 35 degrees . The sulfolane rings were more similar: four of the six molecules exhibit envelope conformations with S-endo, as in molecule B of (II). Despite the conformational differences, the hydrogen-bonding scheme for both molecules is similar: carboxyl -OH groups form hydrogen bonds with carboxyl and sulfone O atoms. Alternating A and B molecules joined by hydrogen bonds between sulfone O atoms and carboxyl -OH groups form parallel chains that extend in the ac plane. Other hydrogen bonds between the carboxyl groups link the chains along the b axis.

The reaction of 2-chloro-5-nitropyridine with two equivalents of base produces the title carbanion as an intermediate in a ring-opening/ring-closing reaction. The crystal structures of the tetra-n-butylammonium salts of the intermediates, C(16)H(36)N(+).C(5)H(3)N(2)O(3)(-), revealed that pseudo-cis and pseudo-trans isomers are possible. One crystal structure displayed a mixture of the two isomers with approximately 90% pseudo-cis geometry and confirms the structure predicted by the S(N)(ANRORC) mechanism. The pseudo-cis intermediate undergoes a slow isomerization over a period of months to the pseudo-trans isomer, which does not have the appropriate geometry for the subsequent ring-closing reaction. The structure of the pure pseudo-trans isomer is also reported. In both isomers, the negative charge is highly delocalized, but relatively small differences in C-C bond distances indicate a system of conjugated double bonds with the nitro group bearing the negative charge. The packing of the two unit cells is very similar and largely determined by the interactions between the planar carbanion and the bulky tetrahedral cation.

The reaction of 2-chloro-3-nitropyridine with two equivalents of hydroxide ion was studied by NMR and X-ray crystallography. On the basis of NMR coupling constants, the originally formed ring-opened intermediate is the pseudo-cis form, as predicted by the SN(ANRORC) mechanism. However, the first intermediate is unstable and isomerizes to a second intermediate, which was isolated. The pseudo-trans geometry of the second intermediate [3-pentenenitrile, 2-nitro-5-oxo, ion(-1), sodium] explains why additional base does not lead to the ring-closing reaction as observed with 2-chloro-5-nitropyridine.

The title five-membered heterocycle, C4H4Cl2OS, adopts an envelope conformation with the S atom at the tip of the flap. All three ring substituents, viz. the sulfoxide O atom and the two Cl atoms, are cis to each other. The two C atoms alpha to the sulfoxide group are also bonded to chlorine. The electron-withdrawing chlorine substituents give rise to weak C-H...O hydrogen bonds with the sulfoxide O atom of a symmetry-related molecule [H...O = 2.44 (2) and 2.61 (2) A, C...O = 3.143 (3) and 3.302 (2) A and C-H...O = 129.9 (19) and 135.1 (19) degrees ]. There is also a possible weak C-H...Cl interaction. Chains of molecules held together by these weak interactions run parallel to the a axis.

Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating into photoreceptors, neurons, and glia. The current-voltage relation of the undifferentiated cells is dominated by a transient inward current that disappears shortly after differentiation. In 20 mM Ba(2+)-containing bath solutions, the current has an activation midpoint near -25 mV and appears to be fully inactivated at -20 mV. Sr(2+) and Ca(2+) are preferred charge carriers relative to Ba(2+), and the current vanishes in the absence of these divalent cations. Cd(2+) blocks the current with an IC(50) of 160 microM, and Ni(2+) blocks in a biphasic manner with IC(50)s of 22 and 352 microM. The current is unaffected when sodium is replaced with other monovalent cations, and it is insensitive to nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. RT-PCR revealed the presence of alpha 1G and alpha 1H mRNA in undifferentiated cells, but following differentiation, a striking reduction of both alpha 1G and alpha 1H mRNA was found, and this was paralleled by the loss of T-type Ca channel currents. alpha 1I subunit mRNA levels were low in undifferentiated and differentiated cells. These results suggest that T-type Ca channels could play a role in undifferentiated retinoblastoma cell physiology since alpha 1G and alpha 1H Ca channel subunit expression is reduced in cells that have differentiated and exited the cell cycle.

We evaluated olfactory attraction of overwintered plum curculio (PC) adults, Conotrachelus nenuphar, to 16 individual volatile components of unripe plum odor in the laboratory using a still-air dual-choice bioassay system and in the field using baited cotton dental wicks attached to boll-weevil traps placed on the ground beneath the canopy of unsprayed apple trees. Two compounds, ethyl isovalerate and limonene, were significantly attractive in both laboratory bioassays and field experiments. In laboratory bioassays, as concentration was decreased across five orders of magnitude, a greater number of compounds elicited responses suggestive of attractancy (except at the lowest concentration). Even so, linalool, 2-hexanone, and 3-hydroxy-2-butanone were the only other compounds showing significant attractiveness in laboratory bioassays, but none of these (nor any other compounds) were significantly attractive in field assays. We suggest that the use of ethyl isovalerate and/or limonene as odor attractants offers potential to increase the efficacy of current traps for monitoring PCs immigrating into fruit orchards during spring.

The aim of this study was to determine whether Escherichia coli lipopolysaccharide at the doses of 25, 50 and 100 microM influences arginine-vasopressin (AVP) secretion in young rat suprachiasmatic nucleus (SCN) neurons. Lipopolysaccharide administered in the medium for 3 h increased significantly the arginine-vasopressin release lasting up to 6 h after treatment. These results provide the first evidence that lipopolysaccharide influences AVP secretion in SCN neurons. Moreover, these findings may explain some central effects observed in vivo after lipopolysaccharide administration.

alpha-Adrenoreceptors are present in the ventricular (VZ) and subventricular (SVZ) zones of the mammalian embryonic forebrain, and contribute towards cell cycle controls in the germinal neuroepithelium [Pabbathi et al., Brain Res. 760 (1997) 22-33.]. Since noradrenaline-containing fibres from the locus coeruleus (LC) and other brainstem nuclei innervate many parts of the CNS from an early stage of embryogenesis, we have used heterochronic cocultures to investigate whether noradrenergic innervation may be the source of trophic support. Dorsal neopallium from E13 rat embryo was cultured in proximity to E15 rostral pons (RP), enabling rapid innervation of the neuroepithelium by differentiated noradrenergic neurons of the LC. The projection of interconnecting fibres into germinal areas of the cortex was established in vitro by retrograde tracing and dopamine beta-hydroxylase (DBH) immunocytochemistry. When functional innervation was prevented by transection of axons connecting the explants, the number of apoptotic (TUNEL-positive) cells in the neopallium was increased. Bromodeoxyuridine (BrdU) double-labelling confirmed that germinal cells were amongst those which underwent apoptosis. When cortical explants were innervated by a source of non-monoaminergic (cholinergic) axons from the E18 basal forebrain diagonal band/septum complex (DS), numbers of apoptotic cells were comparable to those in non-innervated cultures. alpha1 and alpha2 adrenoreceptor antagonists included in the medium of cortical cocultures innervated by noradrenergic axons reversed the survival-promoting effect of innervation. Blockade of alpha1 but not of alpha2 receptors also reduced the numbers of S-phase nuclei in the cortex. The results provide evidence that local delivery of neurotransmitter from embryonic noradrenergic axons terminating in the neocortex can regulate survival and proliferation of neuroepithelial cells.

A component of collagen-tailed acetylcholinesterase (asymmetric; A-AChE) in muscle forms a metabolically-stable pool which can be released from the cell surface only by collagenase, suggesting that part of the enzyme is covalently bound by its tail (COLQ) subunits. We have investigated whether this insoluble pool forms through covalent cross-linking of A-AChE to extracellular matrix glycoproteins by tissue transglutaminase (Tg; type 2 transglutaminase). Tg catalyzed the incorporation of the polyamine substrate 3[H]-putrescine into the collagen tail of affinity-purified avian A12-AChE. Complexes between A12-AChE and cellular fibronectin were also formed in vitro by Tg. In quail myotubes, retinoic acid, which stimulates the formation of epsilon(gamma-glutamyl)lysine isodipeptide bonds by Tg in myotubes, increased the proportion of extraction-resistant (er) A-AChE. Following irreversible inactivation of AChE by diisopropylfluorophosphate, entry of newly-synthesized A-AChE into the extraction-resistant pool was inhibited by a competitive Tg inactivator RS48373-007. The quantity of exogenously-added A 12 AChE incorporated into the extraction-resistant pool in living myotubes was increased by Tg in the presence of calcium. The inhibition of cross-bridge formation in fibrillar collagen by beta-aminopropionitrile, and pre-exposure of myotubes to a monoclonal antibody to fibronectin, resulted in a reduction in the size of the erA-AChE pool present on the cell-surface. The evidence supports the hypothesis that a component of insoluble collagen-tailed AChE, once subject to clustering influences mediated via reversible docking to proteoglycans and their receptors, is anchored at the cell surface through covalent cross-linking by Tg. The high stability of the epsilon(gamma-glutamyl)lysine isopeptide bond is likely to contribute to the observed low turnover of the erA-AChE fraction.

The formation of covalent isopeptide cross-links between cell surface protein molecules by the enzyme transglutaminase C influences cell adhesion and morphology. Retinoid-inducible cross-linking activity associated with this enzyme is present in the developing rat cerebellar cortex [Perry M. J. M. et al. (1995) Neuroscience 65, 1063-1076]. A monoclonal antibody was used to localize transglutaminase C to granule neurons in the developing cerebellar cortex. The enzyme was inducible by retinoic acid both in granule neurons cultured from postnatal rat cerebellar cortex and in cells of the embryonic dorsal rhombic lip, which contain granule neuron precursors. A possible biological function for transglutaminase activity was investigated in living granule neurons, cultured on a biomatrix substratum, studied by time-lapse cinematographic analysis using the transglutaminase inactivator RS-48373-007. Inhibition of cross-linking activity did not influence the number of neurites formed by granule neurons, but caused the destabilization of neurites during the initial outgrowth period, seen as an increase in the number of growth cone retractions and the onset of premature axon collateral formation (bifurcation). Inactivation of cross-linking activity prevented the formation of fascicles between neurites only when cells were cultured on a biomatrix surface. Two glial proteins involved in cell-extracellular matrix interactions, midkine and galectin-3, were identified as putative substrates for granule neuron transglutaminase. The results suggest that covalent cross-link formation by transglutaminase C or a related enzyme generates multimeric molecular forms of glial-derived proteins, and plays a role in stabilizing newly formed neurites. A possible non-pathological role for transglutaminase in the control of axon collateral branching by developing granule neurons in the cerebellar cortex is discussed.

Schwann cells cloned from rat sciatic nerve survive and display self-induced growth suppression, or undergo spontaneous apoptosis, on long-term serum-free subconfluent culture. Strain SCL4.1/F7 sustained the capacity to growth arrest for up to 40 generations. A soluble activity transmitted between neighbouring cells of this strain suppresses DNA synthesis within three cell cycles. Autocrine Schwann cell growth-inhibitory factor (SGIF) operates during the G1 phase of the cell cycle, overcomes the mitogenic action of Schwann cell/serum-associated (platelet-derived growth factor-BB) and axon-associated (axolemma-enriched fraction) stimuli in serum-free conditions, and suppresses DNA synthesis in sciatic nerve Schwann cell cultures in a stage-specific manner. A 35-kDa protein with N-terminal sequence and approximate molecular mass of the C-propeptide of rat alpha1-procollagen I makes a major contribution to SGIF. Growth suppression in the SCL4.1/F7 strain is mediated by the ras/extracellular signal-regulated kinase pathway, is accompanied by down-regulation of erbB2/erbB3 and of tetraethylammonium-sensitive K+ currents, and is followed by transition of cells within 5-10 days from O4+, p75 nerve growth factor receptor (p75NGF-R)+, glial fibrillary acidic protein (GFAP)+ to O4+, p75NGF-R-, GFAP-, periaxin+ phenotypes. Oct-6/SCIP mRNA is present in both proliferating and growth-arrested SCL4.1/F7 cells. These results demonstrate an autocrine/ paracrine loop for the growth arrest of clonally derived Schwann cells in the absence of axons linked in part to the metabolism of collagen. Schwann cells thus appear to self-regulate growth in a negative as well as a positive direction through characterized molecular mechanisms and signal pathways.

The neurofilament L/68 protein (NF-L/68) gene is expressed in the immature Schwann cell phenotype but suppressed after myelin-formation. We have investigated conditions which regulate the activity of the NF-L/68 promoter in green fluorescent protein reporter constructs expressed in the immortal rat Schwann cell strain SCL4.1/F7 in coculture with neurons. Constructs expressed in a plasmid vector containing both the full-length promoter and the 3' proximal 107 bp sequence which includes the cyclic AMP response element (CRE), were active in SCL4.1/F7 cells, but were suppressed as the cells underwent spontaneous growth-arrest. Interaction of SCL4.1/F7 with axons accelerated downregulation of expression from both constructs, however expression of the full-length promoter continued in some cells until the onset of myelin-formation. Expression of the NFL/68 construct recommenced when demyelination was induced in culture by exposure to human sera from patients with paraproteinemic gammopathy. We have demonstrated a method to study the regulation of gene expression patterns in single Schwann cells interacting with neurons and shown that different promoter regions may be controlled by axon-related and -unrelated factors.

Type 2 transglutaminase (Tg), which catalyzes the covalent cross-linking of cytoplasmic proteins during apoptosis, also functions as the alpha subunit of a heterodimeric G-protein (Gh) which can activate phospholipase C-delta1 during the signal transduction pathway linked to alpha1-adrenoreceptors. Continued stimulation of rat forebrain ventricular zone (VZ) germinal cells with the alpha1-agonist phenylephrine during development in vitro suppresses apoptosis and promotes DNA synthesis [Pabbathi et al., Brain Res., 760, 1997, 22-33]. Immunocytochemistry with a monoclonal antibody to Galphah/Tg reveals that alpha1-agonist deprivation during culture of VZ cells in the presence of a protein synthesis inhibitor results after 20 h in a loss of peripheral distribution of the protein and an increase in the reaction product of Tg in the cytoplasm of cells undergoing apoptosis. Using photoaffinity labelling, we observed reduced GTP binding to Galphah/Tg in phenylephrine-deprived cultures. Formation of inositol triphosphate (IP3) and intracellular Ca2+ transients occurred in the presence of phenylephrine. In cultures grown in phenylephrine-deprived conditions in the presence of protein synthesis inhibitor, both the IP3 response and the amplitude and duration of Ca2+ transients were reduced. These results show that loss of signal transduction coincides with the onset of transglutaminase activity in VZ cells during a period when cell survival is reduced following withdrawal of alpha1-agonist, and support the hypothesis that Tg/Galphah could be implicated in both signal transduction and programmed cell death., (Copyright 1998 Elsevier Science B.V.)

We have investigated the possible role of alpha1-adrenoreceptors in regulating the germination of progenitor cells cultured from embryonic rat neocortex. High binding levels of the alpha1-selective radioligand 3[H]prazosin were detected in the forebrain of the rat embryo at E13, and the greatest density of binding sites was localized to the ventricular and subventricular zones. Catecholamine-containing axon terminals were present in these zones in the same period. Germinal neuroepithelial cells retained specific 3[H]prazosin binding in culture. Approximately 25% of cells in culture displayed complex intracellular Ca2+ transients in response to phenylephrine, many of which were abolished with the alpha1B antagonist, chloroethylclonidine. Cultures exhibited concentration-dependent catecholamine stimulation of DNA synthesis mediated by alpha1 receptors in serum-limited conditions. Neuroepithelial cells were labelled via their ventricular processes by intraventricular injection of Fast blue in E13 embryos prior to transfer of the neocortex to dissociated cell culture. Many of labelled cells were present in culture in germinal foci. Some cells which migrated from these foci underwent apoptosis, as determined by TUNEL in situ hybridization. During a transitory period of up to 48 h in culture, alpha1-adrenoreceptor activation by phenylephrine or noradrenaline increased the number of surviving cells. Apoptosis was observed in vivo in both ventricular and subventricular zones of the neocortex from E13 to E15 in increasing numbers. We propose that both the supply of noradrenaline to forebrain germinal cells, and the expression of alpha1-adrenoreceptors on their surface could act to determine whether they die or continue to proliferate.

Inside-out patches containing cGMP-gated channels were excised from catfish rod or cone outer segments and held under voltage clamp. The net cGMP-dependent currents elicited by saturating and subsaturating concentrations of cGMP at +/-30 mV were measured and the dependence of current upon cGMP concentration was determined. The apparent affinity of the channel for its ligand was estimated by fitting these data with the Hill equation. The concentration of cGMP required to give half the maximum current (K1/2) in rod and cone channels at +30 mV was approximately 28 microM and approximately 37 microM, respectively, and was weakly voltage dependent. Thus, cone channels have an intrinsically higher K1/2 than rod channels. For both types of channel, the Hill coefficient was approximately 2.3. In the presence of calcium-calmodulin, the apparent affinity of the rod channel for cGMP decreased by about twofold, but the apparent affinity of the cone channels was unaffected. These results indicate that the open probability of the cone channel for its ligand cannot be modulated by calmodulin. This represents the first significant departure between rod and cone photoreceptors in mechanisms used by phototransduction and suggests that the beta subunit of the cone channel must be different from that of the rod channel.

Tetraalkylammonium compounds and other organic cations were used to probe the structure of the internal and external mouths of the pore of cGMP-gated cation channels from rod and cone photoreceptors. Both rod and cone channels were blocked by tetramethyl- through tetrapentylammonium from the intracellular side in a voltage-dependent fashion at millimolar to micromolar concentrations. The dissociation constant at 0 mV (KD(O)) decreased monotonically with increasing carbon chain length from approximately 80 mM (TMA) to approximately 80 microM (TPeA), where the dissociation constant in rod channels is approximately 50% that of cone channels. N-Methyl-D-glucamine and the buffer Tris also blocked the cone channel in a voltage-dependent fashion at millimolar concentrations, but with lower affinity than similarly sized tetraalkylammonium blockers. Block by tetrahexylammonium (THxA) was voltage-independent, suggesting that the diameter of the intracellular mouth of these channels is less than the size of THxA but larger than TPeA. The location of the binding site for intracellular blockers was approximately 40% across the voltage-drop from the intracellular side. The addition of one carbon to each of the alkyl side chains increased the binding energy by approximately 4 kJ mol-1, consistent with hydrophobic interactions between the blocker and the pore. Cone, but not rod, channels were blocked by millimolar concentrations of extracellular TMA. The location of the extracellular binding site was approximately 13% of the voltage drop from the extracellular side. In cone channels, the two blocker binding sites flank the location of the cation binding site proposed previously.

We have investigated the activity of the Ca(2+)-dependent apoptosis-related transglutaminase type 2 in the mnd/mnd mouse mutant. Transglutaminase activity in mnd/mnd central nervous system (CNS) tissue homogenates was identical to that of healthy animals at 3 months of age, but at 8 months it was greater in the mnd/mnd CNS by up to four times, depending on the region. Western blot analysis showed no difference in the level of immunoreactive transglutaminase type 2 in spinal cord homogenates between mnd/mnd and healthy mice. However, a greater number of acyl donor protein substrates of transglutaminase were identified in mnd/mnd tissue. N epsilon (gamma-Glutamyl)lysine cross-linked product of transglutaminase activity was localized to the soma of degenerating motor neurons in the mnd/mnd mouse spinal cord. We conclude that neurodegeneration in the mnd/mnd mouse is accompanied by activation of transglutaminase at substrate level. Possible mechanisms of activation and its implications for cellular pathology are discussed.

The ability of the divalent cations calcium, magnesium, and barium to permeate through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions and by measuring their ability to block current carried by sodium when presented on the cytoplasmic or extracellular side of the channel. Current carried by divalent cations in the absence of monovalent cations showed the typical rectification pattern observed from these channels under physiological conditions (an exponential increase in current at both positive and negative voltages). With calcium as the reference ion, the relative permeabilities were Ca > Ba > Mg, and the chord conductance ratios at +50 mV were in the order of Ca approximately Mg > Ba. With external sodium as the reference ion, the relative permeabilities were Ca > Mg > Ba > Na with chord conductance ratios at +30 mV in the order of Na >> Ca = Mg > Ba. The ability of divalent cations presented on the intracellular side to block the sodium current was in the order Ca > Mg > Ba at +30 mV and Ca > Ba > Mg at -30 mV. Block by external divalent cations was also investigated. The current-voltage relations showed block by internal divalent cations reveal no anomalous mole fraction behavior, suggesting little ion-ion interaction within the pore. An Eyring rate theory model with two barriers and a single binding site is sufficient to explain both these observations and those for monovalent cations, predicting a single-channel conductance under physiological conditions of 2 pS and an inward current at -30 mV carried by 82% Na, 5% Mg, and 13% Ca.

The permeation of monovalent cations through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions. For monovalent cations presented on the cytoplasmic side of the channel, the permeability ratios with respect to extracellular Na followed the sequence NH4 > K > Li > Rb = Na > Cs while the conductance ratios at +50 mV followed the sequence Na approximately NH4 > K > Rb > Li = Cs. These patterns are broadly similar to the amphibian rod channel. The symmetry of the channel was tested by presenting the test ion on the extracellular side and using Na as the common reference ion on the cytoplasmic side. Under these biionic conditions, the permeability ratios with respect to Na at the intracellular side followed the sequence NH4 > Li > K > Na > Rb > Cs while the conductance ratios at +50 mV followed the sequence NH4 > K approximately Na > Rb > Li > Cs. Thus, the channel is asymmetric with respect to external and internal cations. Under symmetrical 120 mM ionic conditions, the single-channel conductance at +50 mV ranged from 58 pS in NH4 to 15 pS for Cs and was in the order NH4 > Na > K > Rb > Cs. Unexpectedly, the single-channel current-voltage relation showed sufficient outward rectification to account for the rectification observed in multichannel patches without invoking voltage dependence in gating. The concentration dependence of the reversal potential for K showed that chloride was impermeant. Anomalous mole fraction behavior was not observed, nor, over a limited concentration range, were multiple dissociation constants. An Eyring rate theory model with a single binding site was sufficient to explain these observations.

Covalent cross-linking of cell surface proteins by the calcium-dependent enzyme transglutaminase C may be implicated in cell-cell interactions and growth regulation. We demonstrate the presence of the enzyme in rat cerebellar cortex during postnatal development. Transglutaminase C was induced in cerebellar granule neurons in culture by retinoic acid, dibutyryl- and 8-bromo-cyclic AMP analogues and by cultivation on a biomatrix substratum. Cyclic AMP analogues stimulated transglutaminase activity in protein synthesis-dependent and -independent phases. The enzyme was distributed at focal adhesion sites on the axon. By calcium-dependent covalent incorporation of the primary amine acceptor substrate, 5-(biotinamido)pentylamine, an increase in the Ca(2+)-dependent cross-linking of at least 11 substrate proteins in the presence of retinoic acid and dibutyryl-cyclic AMP was detected. Of these substrates, a subset was labelled on the surface of living granule neurons. A low-molecular-weight substrate, p18, was tentatively identified as the retinoic acid-inducible neurite-promoting factor, midkine. Transglutaminase-mediated amine incorporation, midkine and isopeptide cross-links were co-localized to axonal adhesion sites. The results provide evidence of transglutaminase C-catalysed protein cross-linking activity in cerebellar granule neurons and its possible implication in cell-substratum interactions.

Binding of the stable melanocortin(4-9) analogue, Org2766 [Met(O2)-Glu-His-Phe-D-Lys-Phe] to cultured rat sciatic nerve Schwann cells was demonstrated using a biotinylated derivative in semiquantitative histochemical and CELISA assays. Org2766 bound to Schwann cells, but not to fibroblasts, and was displaced maximally by unlabeled Org2766, alpha-MSH and ACTH(1-24). Displacement of Org2766 from the binding sites was considerably reduced by N- and C-truncation of the peptide. Specific binding of Org2766 was also demonstrated in the immortal rat Schwann cell line SCL4.1/F7 and was more pronounced in cells displaying a differentiated morphology. Org2766 and alpha-MSH increased cyclic AMP content of Schwann cells but neither stimulated DNA synthesis when applied alone. However, in the presence of a priming (subthreshold) concentration of the mitogen, cholera toxin, Org2766 and alpha-MSH caused a delayed increase in DNA synthesis. Org2766 did not modulate the expression of several differentiation-related Schwann cell markers. However, Org2766 increased immunoreactivity for p75 low-affinity NGF receptor on Schwann cells and evoked the release of neurotrophic factor(s) that synergized with NGF in stimulating neurite outgrowth in rat DRG neurons. The results indicate that Schwann cells are a primary target for the action of Org2766 and provide evidence for an indirect mechanism by which melanocortins might stimulate neurite sprouting in regenerating peripheral nerve axons.

Treatment of neonatal rats with capsaicin causes a 92.4% loss of calcitonin-gene-related-peptide-immunoreactive unmyelinated sensory afferent fibres in the airways epithelium, vascular smooth muscle and perivascular adventitial layer of lung tissue compared with vehicle-treated controls. Rats were administered Sephadex particles i.v. 8-10 weeks after either capsaicin or vehicle treatment at birth in order to induce a granulomatous tissue inflammation, peripheral blood eosinophilia and pulmonary eosinophil invasion [Laycock et al., Int Arch Allergy Appl Immunol 1986;81:363-367]. The animals also exhibited lung hyperreactivity in vitro in response to carbachol and serotonin (5HT). In Sephadex-treated rats, capsaicin pretreatment did not affect the number of inflammatory cells in peripheral blood, the number of eosinophils in lung tissue, or the distribution of eosinophils in the adventitial tissue of blood vessels. Potencies of concentration-related contractures of lung tissue to 5HT and carbachol were increased by 50- to 100-fold in Sephadex-treated animals compared to controls, but in neither group was potency influenced by capsaicin pretreatment at birth. Recruitment and subsequent regional distribution of inflammatory cells in lung tissue and the increase in lung hyperresponsiveness exhibited in this model of asthma do not appear to involve neuropeptides released from primary afferent neurones.

Neonatal rat Schwann cells were cultured for several months with intermittent exposure to the mitogen, cholera toxin, and infrequent passaging to avoid premature transformation. A cell line SCL4.1/F7 was derived following the cloning of one of these long-term cultures by limiting dilution in liquid medium to select for cells capable of continuous proliferation in the absence of mitogen. F7 cells have been passaged 40 times (80-120 generations) over 14 months. Two substrains were identified at passage 20, one of which ,s diploid and the other which has trisomy 7 (t7). The cell line displays a characteristic flattened or crescent-shaped morphology, substratum adhesion which is calcium-dependent in the millimolar range, and pronounced contact-inhibition of growth. Confluent or subconfluent cultures readily cease proliferation and change to a differentiated (stellate/bipolar) morphology through the mediation of an autocrine growth-inhibitory factor. F7 cells grafted into the site of a crush injury in adult rat sciatic nerves remained viable and myelinated host axons. F7 is the first clonally derived diploid immortal Schwann cell line to have been published and should provide a suitable tool for the study of the biochemical and cellular basis of sheath cell-neuron interactions, myelin stabilization in peripheral nerve and Schwann cell growth autoregulation.

The ACTH/MSH melanocortin core peptide sequence possesses neurotrophic properties in peripheral nerve. During functional neuroanatomical recovery after damage to peripheral nerves, Schwann cells play a significant role in facilitating regeneration. Here we employ a modified super-potent alpha-MSH analogue to solubilise alpha-MSH receptor proteins from cultured primary rat Schwann cells. [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]-alpha-MSH photoaffinity labelled proteins from Schwann cells were analyzed by SDS-PAGE followed by autoradiography. The results indicate that the alpha-MSH receptor proteins labelled have a molecular weight of 42-45 kDa. These data are the first to demonstrate solubilisation and characterisation of alpha-MSH receptors from non-melanoma cells.

Enzymes of the transglutaminase family catalyze the Ca(2+)-dependent covalent cross-linking of peptide-bound glutamine residues of proteins and glycoproteins to the epsilon-amino group of lysine residues to create inter- or intramolecular isopeptide bonds. Transglutaminases can also covalently link a variety of primary amines to peptide-bound glutamine residues giving rise to two possibilities; firstly, where the primary amine has two or more amino groups, further catalysis can result in the formation of cross-linked bridges between glutamine residues, and secondly, where the primary amine is a monoamine, glutamine residues are rendered inert to further modification. The products are therefore in the main, homo- or heterodimers, or extensive, metabolically-stable multimeric complexes or matrices. Ca(2+)-dependent transglutaminase activity is present in the mammalian peripheral and central nervous systems and transglutaminase-catalyzed cross-linking of endogenous substrates has been demonstrated in neurons of Aplysia and the mammalian brain. Transglutaminase activity increases in the brain during development, principally owing to the increasing preponderance of glial cell activity. In a few regions including the cerebellar cortex, activity is also high in early development. Cellular transglutaminases occur widely in differentiating cells and tissues in mammals, with more than one transglutaminase frequently associated with a single cell type. The primary protein sequences of three cellular transglutaminases have been fully determined in different species, together with that of a mammalian protein homologue (band 4.2) which shares extensive sequence homologies with transglutaminases, but lacks the active site cysteine residue. The upstream sequences of two mammalian cellular transglutaminase genes (C and K) contain numerous regulatory sites, and an invertebrate transglutaminase, annulin, is spatially regulated within homeodomains. Multiple molecular forms of transglutaminase C and possibly other cellular transglutaminases exist in mammalian brain. The emerging picture is one of a family of cytosolic and membrane-bound proteins central to several regulatory pathways whose functions is to stabilize the cellular and intercellular superstructure in growing organisms. The targeted formation of glu-lys isopeptide bonds between proteins is central to this function. Cytoskeletal proteins, membrane-associated receptors, enzymes in signal transduction pathways and extracellular glycoproteins are candidate substrates as are polyamines, but few cellular proteins have been identified as components of naturally-occurring covalently-bonded matrices. Transglutaminases participate in the programme of neuronal differentiation in some but not all classes of neurone. Both neuronal and non-neuronal expression of transglutaminases may be important for guidance of migrating neurons or growth cones and sustainment of cell shape and coordinates during development.(ABSTRACT TRUNCATED AT 400 WORDS)

We have measured, characterized, and localized calcium-dependent protein cross-linking activity in rat skeletal muscle, and in myotubes cultured independently or in coculture with spinal neurones, catalyzed by the enzyme tissue transglutaminase (tTG). The enzyme activity was present in both motor endplate and endplate-free zones of rat diaphragm muscle. tTG in the endplate zone was more tightly associated with the tissue. This form of association was absent in extracts of peripheral nerve. Cross-linking of endogenous proteins, as measured by the content of epsilon-(gamma-glutamyl)lysine isopeptide, was higher in the endplate than in the nonendplate zone. Cytosolic (C) and particulate (B) forms of tTG were separated by ion-exchange chromatography from both regions of the muscle. In the motor endplate zone, a higher proportion of tightly bound tTG was recovered as a separate (B1) particulate form. Km values for calcium activation of the three forms of tTG were in the range of 5-15 microM. Immunocytochemistry with polyclonal and monoclonal antibodies revealed the enzyme at motor endplates and at contacts between neurites of rat embryo spinal neurones and myotubes in primary cocultures. Appearance of the B1 transglutaminase could be induced by coculturing myotubes of the mouse C2C12 cell line with neurones. The results suggest that tTG is most concentrated and active at the motor endplate.

The distribution of the retinoid-inducible enzyme, tissue transglutaminase (tTG) in developing rat spinal cord was determined by enzyme assay and immunocytochemistry. tTG activity was at its highest in the forebrain in late foetal development. In hindbrain and spinal cord, elevated activity persisted until after birth. In spinal cord only, a second peak of activity occurred during the first week post partum (P3). tTG was associated with both the cytosolic and particulate tissue fractions throughout spinal cord development, but the particulate component was more prominent in the early postnatal period. tTG was more concentrated during this period in the ventral horn, where the particulate-associated enzyme activity was highest. In spinal cord at 3 days post partum, particulate tTG could be solubilized with lubrol-PX, dithiothreitol and potassium thiocyanate. Both soluble and particulate-associated tTG coeluted with guinea-pig liver transglutaminase C by DEAE-sephacel chromatography. The first peak of tTG activity during late foetal life coincided with the transient localization of the enzyme by immunocytochemistry in vascular endothelia throughout the spinal cord. The second peak of activity at 3 days post partum, by which time vascular immunoreactivity was absent, coincided with the occurrence of small numbers of intensely immunoreactive motor neurones in the ventral horn. Immunoreactive motor neurones were seen predominantly at two levels: the lower thoracic segments and lumbar enlargement. The abnormal appearance of many immunoreactive neurones suggested degenerative changes were occurring. tTG was also present in central canal cluster cells from birth onwards. No neuronal immunoreactivity was seen throughout foetal development. A proportion of motor neurones prepared from E15 spinal cord and grown in coculture with spinal cord astrocytes, were immunoreactive for tTG. All immunoreactive neurones showed signs of degeneration. Addition of myotube-conditioned medium (a source of cholinergic differentiation factor, CDF) reduced the proportion of tTG-immunoreactive neurons in the cultures. Schwann cell-conditioned medium (a source of ciliary neurotrophic factor, CNTF) had a similar but less potent effect on the numbers of immunoreactive neurones. The possibility that tTG is a marker for late, but not early-phase programmed cell death in the developing rat spinal cord is discussed in the light of a proposed role for tTG in the mechanism of natural cell death by apoptosis.

Retinoblastoma cells represent pluripotent neural-progenitor cells which, if induced to differentiate, express many features of mature human retinal neurons. Ca channel currents were recorded from isolated, undifferentiated human retinoblastoma Y79 cells in a bath solution containing 20 mM BaCl2 using whole-cell patch-clamp pipettes containing CsCl. The transient, macroscopic currents inactivated with a time constant of about 20 ms at -20 mV and had other properties similar to low-voltage-activated calcium channels described in other cell types: Activation curves fit by the Boltzmann relation had a midpoint of -32 mV and a slope factor of 6.8 mV (-80 mV holding potential) and inactivation curves had a midpoint of -40 mV and a slope factor of 3.7 mV. Non-stationary fluctuation analysis of the currents performed over a 2 kHz bandwidth indicated that each channel contributed 0.44 pA to the macroscopic transient current at -20 mV, suggesting a unitary conductance of about 7 pS.

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

Rat alpha- and alpha-CGRP are substrates for endopeptidase 24.11 in vitro. Cleavage of both peptides occurs at several points, including an unusual substrate recognition site to the amino side of ala36. In alpha-CGRP this resulted in the early formation of val32-gly-ser-glu35, a sequence previously reported to be a component of the eosinophil chemotactic factor of anaphylaxis (ECF-A). The biological activity of this peptide fragment was confirmed by bioassay. Chemotactic activity in other hydrolysis fragments of both alpha- and beta-CGRP was observed. Both alpha- and beta-CGRP could thus serve as precursors to different eosinophil chemotactic peptide fragments. A novel function of endopeptidase 24.11 may be to modify rather than to terminate the biological activity of CGRP peptides.

The sequence of rat alpha-calcitonin gene-related peptide (CGRP-alpha) contains the tetrapeptide eosinophil granulocyte chemotactic factor Val32-Gly-Ser-Glu35. Peptide fragments formed following hydrolysis of rat CGRP-alpha in vitro by endopeptidase-24.11 were identified. The tetrapeptide fragment was generated following cleavage at a substrate recognition site unusual for this enzyme (-Glu-Ala-). Chemotactic activity of rat CGRP-alpha was increased following hydrolysis. Furthermore, rat CGRP-beta, which lacks the tetrapeptide sequence and is completely devoid of chemotactic activity, displayed low but measurable activity after hydrolysis. Val-Gly-Ser-Glu was identified as the principle fragment with chemotactic activity in rat CGRP-alpha. The results show that the chemotactic activity of the neuropeptide rat CGRP-alpha towards eosinophil polymorphonuclear leukocytes is increased following its hydrolysis in vitro by endopeptidase 24.11 through the formation of a previously identified eosinophil chemotactic tetrapeptide.

1. A patch of plasma membrane was excised, in the inside-out configuration, from the outer segment tip of a catfish cone and recorded electrically with a patch pipette. A solution of 118 mM-NaCl was present on both sides of the membrane. 2. With the solution outside the pipette containing a low concentration (typically several micromoles per litre) of cyclic GMP and the membrane potential held at a non-zero level, brief steps of current indicative of the openings of single ion channels could be detected. There was no sign of desensitization to the ligand over a period of tens of seconds. 3. The prominent openings were associated with a conductance near 50 pS and an open-time constant of 0.5 ms or less. There was also an indication of sub-state openings. 4. The conductance of the large openings appeared to be invariant between -50 mV and +50 mV. However, the macroscopic current-voltage relation measured at a saturating concentration of cyclic GMP showed a slight upward curvature, which we attribute to a voltage dependence in the open probability of the fully liganded channel. 5. The relation between mean current and cyclic GMP concentration had an average Hill coefficient of about 2.4. The Hill coefficient was not affected by membrane voltage, but the conductance was activated by cyclic GMP slightly more readily at depolarizations; this could be adequately explained by a higher open probability of the fully liganded channel at positive voltages. 6. In several experiments, the membrane patch apparently contained a single cyclic GMP-activated channel, in that the measured current never rose above that for a single channel even at high concentrations of cyclic GMP. In these cases, a high concentration of the ligand simply engaged the channel in a literally continuous burst of openings, with an open probability of 0.8-0.9 at between -30 mV and +30 mV. The amplitude distribution of the burst under these conditions could be described by a beta distribution, consistent with the channel switching predominantly between a single closed state and a single open state when fully liganded. 7. Estimates of channel density on the cone membrane ranged from about 2 to 130 microns -2, with an average of 20 microns -2. This observed density is about ten times lower than the density of the homologous channel on rod membrane, being roughly in inverse relation to the tenfold larger surface membrane area of the cone outer segment.(ABSTRACT TRUNCATED AT 400 WORDS)

Myotubes prepared from the Japanese quail embryo at 9 days gestation were cultivated in the presence of glycyl-L-glutamine (Gly-Gln, beta-endorphin C-terminal dipeptide) or glycyl-glutamic acid (Gly-Glu), and changes in the activity of acetylcholinesterase (AChE) molecular forms and binding of 125I-alpha-bungarotoxin (alpha BGT) to cell surface nicotinic acetylcholine receptors were measured. The A12 oligomer was the major form of AChE in the cultures. The activity of all molecular forms of the enzyme was increased in the presence of Gly-Gln, but Gly-Glu did not alter AChE activity. In cells infected with the temperature-sensitive mutant, La31C, of Rous sarcoma virus (ts-RSV) and transferred to the nonpermissive temperature, the A12 form of AChE was absent, but its activity could be induced following exposure of the cells to Gly-Gln. When cells treated in this way were incubated in the presence of collagenase, there was a small but significant loss of A12 AChE activity, indicating that Gly-Gln stimulated the activity of a pool of this oligomer which was mainly but not entirely intracellular. Neither Gly-Gln nor Gly-Glu influenced 125I-alpha BGT binding after exposure of the cells to the peptides for any duration. Neither Gly-Gln nor Gly-Glu influenced the accumulation of cyclic AMP in the cultures. beta-Endorphin is one of a family of peptides that coexist transiently with acetylcholine in lower motoneurones of vertebrates in the perinatal period. This report provides evidence for the selective trophic activity of one of its derivatives toward the postsynaptic cholinergic system in avian muscle cells.

In rat embryo skeletal myotubes, acetylcholinesterase is present, as multiple forms, and can be detected in deposits at the cell surface. Myotubes cultured in the presence of beta-endorphin, exhibit an increased predominance of the globular (precursor) forms of the enzyme, which are largely restricted to intracellular sites associated with nuclei. In the presence of beta-endorphin-(1-27), the relative proportions of the different forms of acetylcholinesterase is similar to that seen in the controls, but the enzyme is intracellular and has a characteristic focal localisation in the myotube.

Beta-endorphin was detected by immunocytochemistry in motoneurons from mouse embryo spinal cord enriched by differential sedimentation and plated onto cultures of embryonic astrocytes from appropriate (cord) and inappropriate (cerebral cortex) sources. Growth of motoneurons on astrocytes for 5 d in vitro improved cell differentiation and suppressed beta-endorphin immunoreactivity. Immunoreactivity was suppressed to a greater degree in motoneurons plated onto cortical rather than spinal cord astrocytes. Some small, dense spinal cord cells also exhibited beta-endorphin immunoreactivity, but staining was unaffected by cultivation on astrocytes. Cell contact and/or hormonal factors deriving from glia may contribute to the regulation of beta-endorphin, a trophic neuropeptide conditionally expressed in normal and pathologic motoneurons.

Nearly thirty growth and trophic factors that have been purified from mammalian tissues in the last 15 yr have been found to share chemical identity. The results of their chemical purification and molecular cloning show that they are two distinct polypeptides (Mr 17,400 and 18,400), each of which gives rise to families of smaller size peptides. These peptides share a common affinity for heparin. In view of this property, a common nomenclature for the two principle peptide growth factors (heparin-binding growth factor classes 1 and 2; HBGF-1 and -2) has been proposed. However, the names acidic and basic Fibroblast Growth Factors (aFGF,bFGF), which were applied to them originally to describe their mitogenic activity, are more commonly in use and will therefore be adopted in this review. Brain tissue is one of the richest sources of FGFs. It has been used as a starting point for their chemical purification and to prepare genomic libraries for molecular cloning of the aFGF and bFGF genes. There is increasing evidence that these growth factors, expressed in neurons and glia throughout the mammalian nervous system, are implicated in neuronal cell proliferation, differentiation, and histogenesis. FGFs have a strong affinity not only for heparin, but also for the related heparan sulphate proteoglycans that are abundant in neural tissues. This fact provides a clue to the importance of tissue-associated proteoglycans in mediating the release, sequestration, and activation of FGFs and the modulation of their receptor binding and bioactivity. The relevance of FGFs to neural development and their mechanisms of action in neurons will be considered in light of the existing literature describing their biological properties and activity in mesodermal cell types. Evidence is reviewed showing that FGFs have in vivo biological activity, ameliorating the degeneration of central and peripheral neurons after axotomy. The presence and implications of high levels of FGFs in adult mammalian brain provides a direction for future research into neural regeneration. The bioactivity of FGFs in neural tissue may not depend on the regulation of their expression per se, but on the subregional modification of their interaction with proteoglycans.

The mass receptor potential of the excised, superfused retina of the bullfrog was studied. Photoreceptor responses were isolated by addition of sodium aspartate to the Ringer solutions. Responses of the cones were monitored independently from responses of the rods by employing a two-flash method of stimulation which took advantage of the very different rates of rapid dark adaptation of rods and of cones. Stimulation with paired flashes of white light at regular intervals caused enhancement of rod response amplitude in that the response grew larger with subsequent flashes until reaching a stable plateau. The degree of enhancement was directly proportional to the amount of light exposure and increased with either increasing stimulus intensity or decreasing stimulus interval. Only the rod response was affected; the cone response was not enhanced by continued stimulation. The effects of 12.5 microM PbCl2 on rod response amplitude were complex. There was a small (less than 10%) but consistent depression of rod response amplitude even when the rods were in the unenhanced state. However, the most striking effect of lead was on the enhanced response, where treatment with 12.5 microM PbCl2 led to a depression of about 33%. When added prior to light stimulation, lead significantly decreased the degree to which the rod response could be enhanced, but never prevented enhancement entirely. Removal of lead resulted in a very large increase in the degree to which the rod response was enhanced by light, even when compared to the first, lead-free control. The cone response was unaffected by 12.5 microM PbCl2.

Isolated, superfused bullfrog retinas were stimulated in such a manner as to allow examination of the dynamics of the shift in the stimulus-response curve that accompanies light adaptation of cone photoreceptors. Observed phenomena were highly temperature dependent with complex Arrhenius relationships, suggesting that the mechanisms responsible for the shift involve multiple enzyme systems.

2-Nonanol, 2-heptanol, octyl decanoate, and octyl octanoate were identified from the heads ofTrigona silvestriana workers. When presented at the nest, 2-nonanol, 2-heptanol, and the mixture of the four compounds elicited angular flights, landing, and buzzing of guard bees. Octyl octanoate elicited a weaker response. No response was given to octyl decanoate, to the ether solvent, or to the control volatile, vanillin.

Neurotensin was localized by the indirect immunofluorescence method in developing organotypic cultures made from slices of the lumbar spinal cord of rat embryos (14-17 days gestation). Less than half of the cultures contained fluorescent cells, and in these specific immunofluorescence was confined to groups of neurones situated in the dorsolateral region of the explants. The neurones became morphologically fully developed after 21 days in culture. Their axons grew up to 1.7 mm in length and travelled within fibre bundles which were oriented around the circumference of the explant. The axonal growth patterns of spinal neurotensin-immunoreactive neurones in tissue culture suggest that they may project over relatively long distances in the rat spinal cord in situ.

Immunocytochemical studies have been performed on rat spinal cord and hypothalamus during development, using an antibody to beta-endorphin. Specific immunoreactivity was demonstrated in histological sections of spinal cord, in ventral and dorsal horn cells and nerve fibres, in the meningeal layer, the ependymal lining of the central canal, and in the endothelium of the ventral spinal artery and other blood vessels. beta-Endorphin immunoreactivity was also distributed widely in neurones, central and peripheral nerve fibres, and in non-neuronal cells in cultures of spinal cord tissue explanted from rat embryos 7--10 days before birth. Immunofluorescence disappeared abruptly after the 28th postnatal day in vivo, and in the fourth week of incubation of embryonic spinal cultures. In contrast, cultures of the ventral diencephalic (hypothalamic) region of embryonic brain at the same gestational age showed a characteristically different pattern of beta-endorphin immunoreactivity which persisted for more than 7 weeks. The results provide evidence for the biosynthesis of a beta-endorphin-like peptide in rat spinal cord during development. The biosynthesis terminates at an early stage both in vivo and in vitro, suggesting that control of the biosynthesis is intrinsic to spinal cord tissue and possibly to the peptide-producing cells themselves.