The chemical characterization of melanin contained in substantia nigra of human brain

The pigment of substantia nigra human brain has been extracted by a mild procedure consisting of washes with phosphate buffer, methanol and incubation with SDS-proteinase. Pyrolisis gas chromatography mass spectrometry infrared spectrometry, termogravimetric analysis and elemental analysis were the techniques used for the chemical characterization. An indole moiety bound to a sulfur containing amino acid and to palmitic acid were the main aspects found in the structure. The presence of a 7% inorganic component was observed. This probably contains Fe, Cu, Zn and Cr which are also relevant, for the formation and the role of melanin in substantia nigra neurons. The fatty acid moiety is chemically bound to the indole structure as it was not eliminated by repeated methanol washing. The same situation occurs for the sulfur containing gropu. Considering these data and the most abundant molecules present in substantia nigra the precursor of neuromelanin seems to be a cysteinyl-cethecol, to which is then bound a palmityl group.     

Mechanism of autoxidation of oxyhaemoglobin

Oxyhaemoglobins from erythrocytes of different animals including fish, amphibians, reptiles, birds, mammals and human beings have been isolated by ion-exchange chromatography over phosphocellulose and the comparative rates of autoxidation of oxyhaemoglobin studied. The mechanism of autoxidationin vitro has been elucidated using toad as well as human oxyhaemoglobin. Autoxidation is markedly inhibited by carbon monoxide as well as by anion ligands, namely, potassium cyanide, sodium azide and potassium thiocyanate. The inhibition by anions is in the same order as their strength as nucleophiles, indicating that it is the oxyhaemoglobin and not the ligand-bound deoxy species which undergoes autoxidation. The structure of oxyhaemoglobin is considered to be mainly and determination of the rate of autoxidation with or without using superoxide dismutase and catalase indicates that the initial process of autoxidation takes place by dissociation of to methaemoglobin and superoxide to the extent of 24%. The superoxide thus produced reattacks oxyhaemoglobin to produce further methaemoglobin and hydrogen peroxide. H₂O₂ is a major oxidant of oxyhaemoglobin producing methaemoglobin to the extent of 53%. A tentative mechanism of autoxidation showing the sequence of reactions involving superoxide, H₂O₂ and OH has been presented.     

Purification and characterization of G proteins from human brain: modification of GTPase activity upon phosphorylation

Summary Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G₅₀) and 36,000 (G₃₆), as determined on SDS-gels. G₃₆ was ADP-ribosylated by pertussis toxin. Thus, G₅₀ could represent a G_sα subunit, whereas G₃₆ could be G_iα or G_oα. G₅₀ was phosphorylated by cAMP dependent protein kinase and protein kinase C. G₃₆ was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G₃₆ by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities withras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated.     

Localization of neuropeptide Y-immunoreactive cells and fibres in the brain of the Japanese quail

Summary In the present study, we have demonstrated, by means of the biotin-avidin method, the widespread distribution of neuropeptide Y (NPY)-immunoreactive structures throughout the whole brain of the Japanese quail (Coturnix coturnix japonica). The prosencephalic region contained the highest concentration of both NPY-containing fibres and perikarya. Immunoreactive fibres were observed throughout, particularly within the paraolfactory lobe, the lateral septum, the nucleus taeniae, the preoptic area, the periventricular hypothalamic regions, the tuberal complex, and the ventrolateral thalamus. NPY-immunoreactive cells were represented by: a) small scattered perikarya in the telencephalic portion (i.e. archistriatal, neostriatal and hyperstriatal regions, hippocampus, piriform cortex); b) medium-sized cell bodies located around the nucleus rotundus, ventrolateral, and lateral anterior thalamic nuclei; c) small clustered cells within the periventricular and medial preoptic nuclei. The brainstem showed a less diffuse innervation, although a dense network of immunopositive fibres was observed within the optic tectum, the periaqueductal region, and the Edinger-Westphal, linearis caudalis and raphes nuclei. Two populations of large NPY-containing perikarya were detected: one located in the isthmic region, the other at the boundaries of the pons with the medulla. The wide distribution of NPY-immunoreactive structures within regions that have been demonstrated to play a role in the control of vegetative, endocrine and sensory activities suggests that, in birds, this neuropeptide is involved in the regulation of several aspects of cerebral functions.Abbreviations AA archistriatum anterius - AC nucleus accumbens - AM nucleus anterior medialis - APP avian pancreatic polypeptide - CNS centrai nervous system - CO chiasma opticum - CP commissura posterior - CPi cortex piriformis - DIC differential interferential contrast - DLAl nucleus dorsolateralis anterior thalami, pars lateralis - DLAm nucleus dorsolateralis anterior thalami, pars medialis - E ectostriatum - EW nucleus of Edinger-Westphal - FLM fasciculus longitudinalis medialis - GCt substantia grisea centralis - GLv nucleus geniculatus lateralis, pars ventralis - HA hyperstriatum accessorium - Hp hippocampus - HPLC high performance liquid chromatography - HV hyperstriatum ventrale - IF nucleus infundibularis - IO nucleus isthmo-opticus - IP nucleus interpeduncularis - IR immunoreactive - LA nucleus lateralis anterior thalami - LC nucleus linearis caudalis - LFS lamina frontalis superior - LH lamina hyperstriatica - LHRH luteinizing hormone-releasing hormone - LoC locus coeruleus - LPO lobus paraolfactorius - ME eminentia mediana - N neostriatum - NC neostriatum caudale - NPY neuropeptide Y - NIII nervus oculomotorius - NV nervus trigeminus - NVI nervus facialis - NVIIIc nervus octavus, pars cochlearis - nIV nucleus nervi oculomotorii - nIX nucleus nervi glossopharyngei - nBOR nucleus opticus basalis (ectomamilaris) - nCPa nucleus commissurae pallii - nST nucleus striae terminalis - OM tractus occipitomesencephalicus - OS nucleus olivaris superior - PA palaeostriatum augmentatum - PBS phosphate-buffered saline - POA nucleus praeopticus anterior - POM nucleus praeopticus medialis - POP nucleus praeopticus periventricularis - PP pancreatic polypeptide - PYY polypeptide YY - PVN nucleus paraventricularis magnocellularis - PVO organum paraventriculare - R nucleus raphes - ROT nucleus rotundus - RP nucleus reticularis pontis caudalis - Rpc nucleus reticularis parvocellularis - RPgc nucleus reticularis pontis caudalis, pars gigantocellularis - RPO nucleus reticularis pontis oralis - SCd nucleus subcoeruleus dorsalis - SCv nucleus subcoeruleus ventralis - SCNm nucleus suprachiasmaticus, pars medialis - SCNl nucleus suprachiasmaticus, pars lateralis - SL nucleus septalis lateralis - SM nucleus septalis medialis - Ta nucleus tangentialis - TeO tectum opticum - Tn nucleus taeniae - TPc nucleus tegmenti pedunculo-pontinus, pars compacta - TSM tractus septo-mesencephalicus - TV nueleus tegmenti ventralis - VeL nucleus vestibularis lateralis - VLT nucleus ventrolateralis thalami - VMN nucleus ventromedialis hypothalami A preliminary report of this study was presented at the 15th Conference of European Comparative Endocrinologists, Leuven, Belgium, September 1990     

Exercise performance of normothermic and hyperthermic rats: Effect of warm rearing

1. 1.|Hypothalamic and rectal temperatures were recorded in 8 warm-reared (wr) and 12 control rats. Rats ran to exhaustion at a constant speed of 1.5 km h⁻¹ but at a variable ambient temperature adjusted to stabilize their hypothalamic temperature at 38.0°C (normothermia) or 41.0°C (hyperthermia). Blood lactate concentrations were determined before and after exercise.

2. 2.|Exercise caused exhaustion in normothermic control rats after 62.08 ± 5.43 min and in wr rats after 29.64 ± 2.09 min.

3. 3.|Hyperthermia shortened to one half (to 12.24 ± 1.36 min) and to one fourth (to 16.15 ± 1.20 min) the endurance time in wr and control rats, respectively.

4. 4.|There were no correlations between lactate concentraion and hyperthermia or endurance time.

5. 5.|In conclusion, in rats and other animals which have safe refuges, hyperthermia interferes with the ability to continue exercising.

High-affinity [3H]inositol uptake by dissociated brain cells and cultured fibroblasts from fetal mice

The accumulation of [³H]inositol by mechanically dissociated brain cells and cultured skin fibroblasts from fetal mice was examined. Uptake by both tissues was strongly dependent on temperature and the presence of sodium ions. Brain and fibroblast uptake also responded similarly to inhibition by inositol isomers and phloridzin. At lower concentrations of inositol, both tissues exhibited high-affinity uptake kinetics with apparent K_m values near 30 M, similar to values observed previously in human fibroblasts and other cultured cells. The activity of brain high-affinity uptake was nearly an order of magnitude lower than that of fibroblasts, however, and was in part confounded by the presence of a low-affinity or simple diffusion system operating at inositol concentrations above 100M. Brain preparation from adult mice also showed evidence of high-affinity, Na⁺ dependent uptake, but its activity was significantly diminished relative to that of fetal brain preparations. Our results demonstrate that a high-affinity inositol transport system closely resembling that found in cultured cells is expressed in the developing mouse brain.     

The phylogenic expression of plasmolipin in the vertebrate nervous system

Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phylogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonal antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these data are discussed.Abbreviations EDTA Ethylene diamine N.,NN tetracetic acid - EGTA Ethylene glycol bis-(B-Aminoethyl Ether) N,,NN tetracetic acid - MES ([N-Morpholino] ethane sulfonic acid) DCCD, N, Dicyclohexyl carbodiimide     

Effect of guanidinoethanesulfonic acid on brain monoamines in the mouse

Guanidinoethanesulfonic acid (GES) is known to induce convulsive seizures when administered intracisternally to rabbits and cats. The effects of GES on behavior, electroencephalographic recording and brain monoamine levels were examined in mice. When GES (900 nmol) was intraventricularly injected into mice, focal clonic movements of the face, vibrissae and ears together with twitching of the limbs were observed 0.5–1 min after the injection. Hypersensitivity was observed up to 7 min after the injection, after which the mice behaved normally. GES also induced sporadic spike discharges on electrocorticogram. The levels of 5-hydroxytryptamine (5-HT) of the GES-injected mice were lower than those of the saline-injected mice in the hippocampus, diencephalon, pons-medulla oblongata and cerebellum 5 min after the injection. No changes in the norepinephrine or dopamine levels were found after the GES injection. The level of 5-hydroxyindoleacetic acid increased in the striatum and cerebellum 5 min after the GES injection. These results suggest that GES-induced convulsive activities enhance the serotonergic neuroactivity in order to suppress the convulsions.     

Changes in free amino acid concentrations in serum,brain, and CSF throughout embryogenesis

Using the developing chick embryo as a model and a very sensitive micromethod for amino acid analysis, a complete analysis is presented of the developmental changes in free amino acid concentration in the blood, in the CSF, and in two different brain regions (optic lobe and frontal lobe) of the chick embryo (from day 4 of incubation, until day 5 post hatching). The developmental profile of Lys is the only one that is almost identical in all three compartments. The developmental profiles of the serum and of the brain are very similar for Arg and Phe, less so for Leu and Gly, and towards the end of the embryonic period, similar also for Val, Ile, Trp, and Met. The amino acid concentrations in the CSF are either much lower than in serum and brain already at the earliest stages, or they progressively decline to levels lower than those in brain and serum, most rapidly between day 6 and 8 of embryonic life. The concentrations of neuroactive amino acids (Gln, Glu, Asp, GABA, Tau, and Gly) in both brain regions begin to increase very early, and continue to rise, except Tau, which goes through a maximum at day 8. Comparative analysis of the developmental profiles of each amino acid in serum, brain, and CSF reveals that the blood supply and the cellular uptake, retention, and metabolism by neural cells are the major determinants of the free amino acid pool of the developing brain.     

Lithium-induced decrease of brain inositol and increase of brain inositol-1-phosphate is transient

The effects of a single does of LiCl (2.5 or 10 mEq/kg) on brain inositol and inositol-1-phosphate (Ins1P), intermediates of brain phosphoinositude (PI) turnover, were determinated in male Han: Wistar rats. There was a remarkable, 36–58 fold elevation of brain Li⁺ as the single does of LiCl was increased 4-fold. Moreover, the accumulation of brain lithium was slow during repeated administration of LiCl. Brain lithium did not correlate with changes in brain PI turnover either after a single or repeated doses. Thus, after a single does of LiCl the increases in brain Ins1P were much less than the decreases in brain inositol. Also, brain inositol was significantly decreased only with the high dose of LiCl whereas brain Ins1P accumulation was more prominent with the lower dose. Moreover, repeated daily doses of LiCl only transiently increased brain Ins1P at 1 and 7 d whereas inositol remained at control levels throughout the 14 d observation period. Lithium probably caused the transient decrease in brain inositol by inhibiting several enzymes, in addition to the inhibition of myo-inositol mono-phosphates, in the PI cycle. Moreover, a slow dampening down of PI turnover by lithium, possible via an inhibitory action on G-protein-coupling, may also explain the present findings.