Catecholaminergic contributions to the neuronal machinery of the olfactory bulb: aftoradiographic, immunohistochemical and evoked feild potential studies

aftographic exeperiments on the localization of radiolabelednoradrenaline, dopamine and dopa, as well as immunohistochemicalstudies on hydroxylase-like activity, are summarized and comparedin both rat and turtle olfactory bulbs. Evoked field potentialstudies on effects of dopamine are also discussed. The histochemicalstudies suggest that dopaminergic periglomerular neurons arethe most significant cellular component of the catecholaminergicsystem in the olfactory bulb of both species. Scattered fluorescentcell group was also present in the internal plexiform layerand superficial granule cell layer of the turtle olfactory bulb.Other fibres, not related to intrinsic bulbar neuronal cellbodies, were also labeled, mostly in the granule cell layerbut also in the external plexiform layer. These might belongto a centrifugal catecholaminergic system from brain stem neurons.In the in vitro turtle olfactory bulb, dopamine and apomorphinedepressed the amplitude of field potentials evoked by a singlevolley in the olfactory nerve or lateral olfactory tract, andreduced the depression and latency of reponses when paired volleywere delivered. It is suggested that catecholaminergic systemsplay a key role in modulating mitral cell activity through actionsin both superficial (glomerular) and deep (granule) layers.This may involve direct actions, or other, non-catecholaminergicinterneurons.     

Ntau-methylhistidine excretion is a valid index of myofibrillar protein breakdown in the guineapig (Cavia porcellus).

The recovery of radioactivity in the urine of guineapigs following a bolus intravenous dose of chromatographically pure 14C-Ntau-methylhistidine was measured in order to test whether the excretion of Ntau-methylhistidine (Ntau-MH) is a valid index of myofibrillar protein breakdown in these animals. Four male and four female guineapigs were dosed and after 7 days, 91.65+/-2.82% and 3.58+/-0.91% of injected radioactivity was recovered in the excreta and tissues, respectively. The average total recovery of 95.2+/-3.0% was not significantly different from 100%. Male guineapigs excreted the radioactivity more slowly than females (70% of the dose excreted within 74 h vs 39 h, respectively) but cumulative excretion at 7 days was the same for each sex. Chromatographic analysis of the urine showed almost all of the radioactivity to be associated with a single peak corresponding to Ntau-MH, indicating a lack of significant metabolism. These data show that although the clearance of 14C-Ntau-MH is slower than in rats or humans the urinary excretion of Ntau-MH is a valid index for myofibrillar protein degradation in the guineapig.     

Noradrenaline nerve terminals in the hippocampal region of the rat and the guinea pig

Summary The hippocampal region of the rat has been studied with the histochemical fluorescence method for demonstration of catecholamines and 5-hydroxytryptamine. Noradrenalin nerve terminals of characteristic appearance were observed throughout the region, but the density of the terminals in the various areas differed considerably. The highest amount of terminals were seen in the hilus of the area dentata of the rat. Otherwise, noradrenalin nerve terminals constitute a small percentage of the afferents to the hippocampal region. Noticeable differences existed between rat and guinea-pig. In both species there was a conspicuous lack of stratification and delineation of areas as compared with the pictures seen with most other methods, which have revealed precise architectonic patterns.Supported by a Research Grant (12X-715-02) from the Swedish Medical Research council and by a Grant from Stiftelsen M. Bergwalls Minne.     

Bacteriochlorophyll organization and energy transfer kinetics in chlorosomes from Chloroflexus aurantiacus depend on the light regime during growth

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 mol m^–2 s^–1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the Q_Y band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - DAS decay associated spectrum - PMSF phenylmethylsulfonyl fluoride     

Reconstitution of the DNA base excision—repair pathway

Background: The base excision–repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision–repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision–repair, we have attempted to reconstitute the pathway in vitro using purified enzymes.Results We report here the successful reconstitution of the base excision–repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5′ terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5′ nuclease function of DNA polymerase I.Conclusion Repair of uracil in DNA is achieved by a very short-patch excision–repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.     

Rat liver foci study on coexposure with 50 Hz magnetic fields and known carcinogens

A study was performed to investigate possible interactions by magnetic fields (MF) with the processes of initiation and promotion of chemically induced preneoplastic lesions in rat liver. Male Sprague-Dawley rats were subjected to a 70% partial hepatectomy followed after 24 h by i.p. injection of diethylnitrosamine (DENA) as a tumour initiator. Starting one week after the DENA-treatment phenobarbital (PB) was given to promote growth of enzymatically altered foci of liver cells. MF was applied immediately after the partial hepatectomy and continued until sacrifice after 12 weeks of PB exposure. Homogenous horizontal AC magnetic fields with a frequency of 50 Hz and flux densities of 0.5 μT or 0.5 mT were used. The rats coexposed with MF and DENA plus PB did not gain weight as much as the rats exposed to the chemical agents only. The MF-exposure also resulted in a slight reduction in size and numbers of the focal lesions. The results suggest an interaction of MF with the processes of chemical carcinogenesis either as a result of stress or depending on effects on the proliferation of preneoplastic cells. © 1993 Wiley-Liss, Inc.     

Laminin and Neuropeptide Y Are Increased by Synapsin Transfection in Cultured NG108-15 Neuroblastoma/Glioma Hybrid Cells

Abstract: We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite outgrowth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E₁ plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation.     

Calbindin D-28k-immunoreactivity in rat muscle spindles during postnatal maturation and after denervation

Summary Calbindin D-28k-immunoreactivity has been demonstrated in some of the intrafusal muscle fibres and in the capsule of adult rat muscle spindles. In this study, the immunocytochemical localization of calbindin D-28k in the muscle spindles of triceps surae muscle was studied during postnatal maturation and after denervation. In young rats calbindin D-28k-immunoreactivity was seen in a few intrafusal fibres, first at the age of 4 days. At the 7th day, three calbindin D-28k-immunoreactive fibres and one unlabelled fibre were seen in most muscle spindles, as in adult rats. The spindle capsule and perineurial sheath of nerves were first seen to exhibit calbindin D-28k immunoreactivity at the age of 14 days, and thereafter the localization of calbinding D-28k-like immunoreactivity was similar to that in adult rats. After denervation, calbindin D-28k-immunoreactivity remained in intrafusal muscle fibres and the spindle capsule for a long period. After two months of denervation, calbindin D-28k immunoreactivity could still be seen in the spindle capsule, but the intrafusal fibres were not labelled.The innervation is known to have trophic effects on the intrafusal fibres. The present findings suggest that the expression of calbindin D-28k-immunoreactivity in maturating muscle spindles may be induced by the developing innervation. The decrease of calbindin D-28k-immunoreactivity in intrafusal fibres after denervation may be due to the loss of trophic factors released by the nerves.     

A rapid method for the analysis of N tau-methylhistidine in human urine

A novel, automated method is described for the determination of N^τ-methylhistidine in human urine. The method uses a modification (H. Nakamura and J. J. Pisano (1976) Arch. Biochem. Biophys.177, 334–335) of the reaction of fluorescamine with amines, which renders it specific for certain imidazoles. Interference due to histidine and histamine is selectively removed by prior reaction with aldehydes. The fluorescence yield for N^τ-methylhistamine is 280, 440, 50, and 1.7, respectively. The concentrations of N^τ-methylhistidine in human urine as determined by this technique correlate well (r = 0.99) with those determined by ion-exchange chromatography. Furthermore, the technique is rapid (6–7 samples/h net throughout), is reproducible (coefficient of variation 1.8%), requires no prior treatment of the sample, and is implemented with widely available equipment.