Localization of substance P-like immunoreactive fibers in the thoracic spinal cord of guinea pig

Summary A dorsal-horn fiber system is revealed in the thoracic spinal cord of guinea pig by means of substance P immunocytochemistry. This system has repeated craniocaudal and/or caudo-cranial extensions and possesses five main components: (1) a superficial network, situated beneath the dorsolateral surface of the spinal cord. This network is connected with the dorsal root fibers and the accumulations of substance P-like immunoreactive (SP-LI) fibers in the Lissauer's tract; (2) an accumulation of SP-LI fibers in the Lissauer's tract at the border of the dorsal horn; (3) two collateral SP-LI fascicles (one lateral and one medial) emerging from the SP-LI fiber accumulation in the Lissauer's tract; (4) a transversal fascicle running through laminae III–V, and (5) an SP-LI network in the region of the lateral spinal cord nucleus. These components of the dorsal-horn fiber system show widespread connections with ipsi-and contralateral spinal cord areas, connecting them in cranio-caudal and/or caudo-cranial directions. The SP-LI dorsal-horn system has close relationship with groups of preganglionic sympathetic cells in the intermediate zone of the spinal cord, respective with the vegetative network of this zone. It is suggested that some fibers of the dorsal-horn system that originate from dorsal-root ganglia may represent primary sensory or visceral afferents. It is likely that the dorsal-horn fiber system and the vegetative network of the thoracic spinal cord may represent the morphological basis for the integration of (1) the central and peripheral vegetative nervous systems, and (2) the somatic and vegetative nervous system.     

Reduced pyridine nucleotides in Pseudomonas carboxydovorans are formed by reverse electron transfer linked to proton motive force

In cell suspensions of Pseudomonas carboxydovorans pulsed with lithotrophic substrates (CO or H₂) in the presence of oxygen, formation of reduced pyridine nucleotides and of ATP could be demonstrated using the bioluminescent assay. Experiments employing base-acid transition, an uncoupler and inhibitors of ATPase or electron transport enabled us to propose a model for the formation of NAD(P)H in chemolithotrophically growing P. carboxydovorans.The protonophor FCCP (carbonly-p-trifluormethoxyphenylhydrazon) inhibited both, formation of NAD(P)H and of ATP. In the absence of oxygen, a chemical potential imposed by base-acid transition resulted in the formation of NAD(P)H and ATP when electrogenic substrates (CO or H₂) were present. This suggests proton motive force-driven NAD(P)H formation. The proton motive force was generated by oxidation of substrate, and not by ATP hydrolysis, as obvious from NAD(P)H formation during inhibition of ATP synthesis by oligomycin and N,N-dicyclohexylcarbodiimide.That the CO-born electrons are transferred via the ubiquinone 10-cytochrome b region to NADH dehydrogenase functioning in the reverse direction, was indicated by inhibition of NAD(P)H formation by HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) and rotenone, and by resistance to antimycin A.We conclude that in P. carboxydovorans, growing with CO or H₂, electrons and a proton motive force, generated by respiration, are required to drive an reverse electron transfer for the formation of reduced pyridine nucleotides.Abbreviations CODH carbon monoxide dehydrogenase - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl-p-trifluormethoxyphenylhydrazon - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - pmf proton motive force     

Partial purification,subunit structure and thermal stability of the photochemical reaction center of the thermophilic green bacterium Chloroflexus aurantiacus

Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides (M_r = 30000 and 28000) in a reaction center complex of M_r = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile.     

Studies on the complex formed between glucagon and dicaprylphosphatidylcholine

The interaction between glucagon and dicaprylphosphatidylcholine (DCPC) was studied by fluorescence, circular dichroism and calorimetry, as well as by ¹H- and ³¹P-nuclear magnetic resonance. The water-soluble lipid-protein complex was also characterized by gel filtration and ultracentrifugation. The complex appeared to be monodisperse by sedimentation equilibrium measurements, with a molecular weight of (4.55 ± 0.57)·10⁴. This complex contained approximately 7 molecules of glucagon and 35 molecules of phospholipid. Proton-decoupled ³¹P-NMR spectra of the phospholipid in the lipid-protein complex display narrower resonances than those of sonicated vesicles of DCPC, and ¹H-³¹P coupling could be detected in proton coupled spectra. These NMR results, together with gel-filtration results, suggest that glucagon ‘solubilizes’ phospholipid aggregates, forming a lipid-protein complex which is smaller than sonicated preparations of DCPC. ¹H-NMR resonance of both the methionine methyl group (met-27) and the aromatic envelope of glucagon are broadened by the phospolipid, indicating that the C-terminal region and the aromatic residues are involved in the interaction with the phospholipid. Nuclear magnetic resonance titrations of the imidazole ring C(2) and C(4) protons of the histidine residue of glucagon show that DCPC lowers the pK of the imidazole. The alterations caused by the phospholipid in the far and near ultraviolet CD spectra of glucagon reflect, respectively, the increased helix content of the hormone and the fact that the aromatic residues are located in a more structured environment. The phospholipid also alters the fluorescence properties of glucagon, shifting the fluorescence emission maximum of the hormone to shorter wavelength, and enhancing its relative intensity. This suggests that the fluorophore is experiencing a more hydrophobic environment in the presence of the lipid. Binding of glucagon to the phospholipid was analysed by Scatchard plots of the enhancement of fluorescence caused by the phospholipid and showed that the equilibrium binding constants of glucagon to DCPC are (4.4 ± 0.5)·10⁴M^?1 and (7.5±0.5)·10⁴M^?1, at 15°C and 25°C, respectively. The average number of moles of phospholipid bound per mole of glucagon is 4.4±0.6. The isothermal enthalpy of reaction of glucagon with DCPC is ?20.5 kcal/mol of glucagon at 25°C and ?32.5 kcal/mol of glucagon at 15°C. The observed enthalpies can arise from glucagon-induced cyrstallization of the phospholipid, from the non-covalent interactions between the peptide and lipid as well as from the lipid-induced conformational change in the protein. These results demonstrate that, unlike the complexes formed between glucagon and phospholipids which form more stable bilayers, the complex formed between glucagon and DCPC is stable over a wide range of temperatures, including temperatures well above the phase transition.     

Trapping, loss and annihilation of excitations in a photosynthetic system: II. Experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata

In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, k_h, and the rate of trapping by an open reaction center, k^o_t, can be estimated. For R. rubrum it is found that λ = 14−17, k_h = (1−2)·10¹² s⁻¹ and k^o_t = (4−6)·10¹¹ s⁻¹, for Rps. capsulata λ ≈ 30, k_h ≈ 4·10¹¹ s⁻¹ and k^o_t ≈ 3·10¹¹ s⁻¹. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria.     

On the O2 flash yields of two cyanophytes

We explored O₂ flash yield in two cyanophytes, Anacystis nidulans and Agmenellum quadruplicatum. On a rate-measuring electrode, a single flash gave a contour of O₂ evolution with a peak at about 10 ms which was maximum (100) for 680 nm background light. On 625 nm illumination the peak was smaller (62) but was followed by an increased tail of O₂ attributed to enhancement of the background. After a period of darkness, repetitive flashes (5 Hz) gave a highly damped initial oscillation in individual flash yields which finally reached steady state at 94% of the yield for 680 nm illumination. When O₂ of repetitive flashes was measured as an integrated flash yield the results was distinctive and similar to that for a continuous light 1 (680 nm). An apparent inhibition of respiration which persisted into the following dark period was taken as evidence for the Kok effect. With a concentration-measuring electrode, integrated flash yield vs. flash rate showed the same nonlinear behavior as O₂ rate vs. intensity of light 1. We draw three conclusions about the two cyanophytes. (a) The plastoquinone pool is substantially reduced in darkness. (b) Because of a high ratio of reaction centers, reaction center 1 / reaction center 2, for the two photoreactions, saturating flashes behave as light 1. (c) Because repetitive flashes are light 1, they also give a Kok effect which must be guarded against in measurements designed to count reaction centers.     

Properties of narrow-range ampholytes isolated from wide-range ampholyte preparations

A simple procedure for obtaining useful narrow-pH-range ampholytes from inexpensive laboratory-synthesized ampholytes by preparative isoelectric focusing in Pevikon is described. The narrow range ampholytes prepared in this way are comparable to commercial ampholyte preparation as judged by conductivity, buffer capacity, pH gradient formation, and resolving power. These inexpensive narrow-range ampholytes are particularly well suited to preparative isoelectric focusing applications requiring large quantities of ampholytes.     

Multiple Forms of Choline-O-Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.     

24-hour variation in content and release of hypothalamic neuropeptides in the rat

The tissue content of up to eight neuropeptides, viz bombesin (BOM), cholecystokinin (CCK-8), neurotensin (NT), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), somatostatin (SRIF), substance P (SP) and vasoactive intestinal polypeptide (VIP), in rat hypothalami removed at various times of the day, was measured using specific radioimmunoassays. There was significant variation in the content of BOM, CCK-8, NT, PHI, SP and VIP across a 24-h period. The levels of BOM, CCK-8 and NT were lowest around the onset of darkness (1900 h) and rose throughout the night to reach a peak around the time of lights on. Hypothalamic content of all eight peptides fell between 0700 h and 1300 h by an average of 45 +/- 4%. Basal release of these peptides, as well as that in the presence of 48 mM potassium (K+), was measured from hypothalami removed between 0700 and 1900 h and incubated in vitro in a CSF-like medium. Basal secretion of NT significantly increased, whilst that of CCK-8 significantly decreased over the same period. There was no significant change in the basal release of the other neuropeptides. The release in the presence of 48 mM K+ of SP decreased significantly during the day, whilst that of VIP significantly increased. There was also a significant change in the stimulated release of BOM, levels falling during the morning and rising again at 1900 h. 48 mM K+ caused a significant increase in the release of SRIF and SP at all times tested. Whilst 48 mM K+ induced a significantly higher release of CCK-8 and NT in the morning, this stimulus was ineffective in the evening. The contrary was true in the case of BOM, NPY and VIP, where a significant stimulation was induced only at 1900 h. The possible implications of these findings are discussed.     

Modulation of locomotor activity by substance P in rats

The effects of intraperitoneally or intracerebrally (DA A-10 area) administered substance P (SP) on locomotor activity of rats were studied in an exact 12-h light/12-h dark cycle changing from dark to light at 6 a.m. SP was administered either at 11 a.m. (light phase, minimal locomotor activity) or at 7 p.m. (dark phase, maximal locomotor activity). The effects of 12.5 micrograms/kg SP intracerebral and 125 micrograms/kg SP intraperitoneal were very similar. In the light phase SP produces excitation but inhibition of locomotion in darkness. Hence, the effect of SP depends on the internal mechanisms controlling motor activity and tends to level off the spontaneous circadian oscillation. We found a long lasting SP effect during both the light and dark period. The present experiments led us to the conclusion that SP has a levelling effect on locomotor activity. Probably this effect might be explained as SP's action on the dopaminergic pathway or dopamine metabolism, because the dopamine content in neurons also has a circadian rhythm.