Cloning and sequence analysis of cDNAs encoding the MHC class II beta chain in Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) cDNAs encoding the major histocompatibility complex (Mhc-Sasa) class II beta chain were isolated from a leucocyte library by a polymerase chain reaction (PCR) approach. Three different cDNAs (c144, c22, and c157) encoding the entire mature beta chain have been analyzed. Clone c144 differs from clone c157 in 12.6% of the nucleotides in the beta 1-encoding region. The corresponding differences between clones c144 and c22, and clones c22 and c157, are 10.3% and 5.2%, respectively. This variation is, at least in part, most likely attributable to allelism. The similarity indices between the highly conserved beta 2 domains from Atlantic salmon and corresponding sequences from humans (DQ beta), chicken (BL beta), carp (TLAII beta-1), and rainbow trout (O.M. No. 55) are 45%, 40%, 66%, and 97%, respectively. Variable residues in the beta 1 domains from Atlantic salmon correspond with polymorphic sites of beta 1 domains from higher vertebrates. The frequency of substitutions in the beta 1-encoding region exceeds that in the 3'-untranslated (UT) region with several folds, indicating extensive beta 1 polymorphism in Atlantic salmon.

Differential expression of I alpha- and II alpha-globin genes in Norwegian dairy goats.

Fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis were used to determine the ratio between I alpha- and II alpha-globin chains in Norwegian dairy goats. Three different phenotypes, designated normal (N) with I alpha- to II alpha-globin ratio 3:1, reversed (R) with ratio 1:2 and double-reversed (RR) with no I alpha-globin, were described. Family studies indicated that the R animals were heterozygous, and the RR animal homozygous, for a haplotype without a functional I alpha-globin product. Southern blot analysis of goat DNAs digested with six different restriction enzymes showed that the different ratios of alpha chain expression could not be due to a deletion of the I alpha-gene and/or duplication or triplication of the II alpha-globin genes. The homozygous reversed animal with no detectable I alpha-globin had a mild anaemia.

Postnatal development of glutamatergic, GABAergic, and cholinergic neurotransmitter phenotypes in the visual cortex, lateral geniculate nucleus, pulvinar, and superior colliculus in cats.

We have analyzed the postnatal development of glutamatergic/aspartergic, GABAergic, and cholinergic neurotransmitter systems in the visual cortical Areas 17 and 18, lateral geniculate nucleus (LGN), pulvinar, and the visual and non-visual parts of superior colliculus (SC) in kittens. High-affinity uptake of D-aspartate (HA D-Asp), glutamate decarboxylase (GAD), and choline acetyltransferase (ChAT) activities were measured as a means of probing the development of the respective transmitter systems. HA D-Asp exceeded the adult level several-fold in all areas during the postnatal period which corresponded with the period of maximal dendritic/axonal branching patterns and synapse densities in the respective regions. GAD exhibited a gradual increase towards adult levels during the first month. The adult level was reached during postnatal week (PNW) 5-6 in Areas 17 and 18, during PNW3 within LGN, pulvinar, and the visual part of SC. In the nonvisual part of SC, the adult GAD level was reached as early as PNW2. ChAT exhibited biphasic developmental profiles in Areas 17 and 18. An initial peak of near adultlike activity in PNW2 was followed by a decline and subsequently by a slow increase towards adult levels during PNW5-17. ChAT developed very slowly in LGN and pulvinar, and in the latter structure only approximately 70% of the adult activity had been attained by PNW17. In both subdivisions of SC, ChAT had reached adult levels during PNW3-5. Dark-rearing from birth until PNW6 moderately attenuated GAD development in all areas and increased ChAT activity in Areas 17 and 18 but did not affect development of HA D-Asp in any part of the kitten visual system. Our neurochemical findings in the developing cat visual system are consistent with available evidence regarding localization of neurotransmitter systems, as well as postnatal changes in terms of cytoarchitectonics, synaptogenesis, functional development, and susceptibility to neonatal dark-rearing in visual pathways.

The acute effect of ammonium acetate on levels of amino acids in the intact and decorticated rat neostriatum.

Unilateral frontal cortex ablations were performed in rats so that the glutamate terminals in the ipsilateral rostral neostriatum were removed. At 1 or 7 days later, intraperitoneal injections of ammonium acetate induced different changes in amino acid concentrations in the intact and deafferentated neostriatum. After 1 day, the level of glutamate decreased only in the intact side, whereas that of glutamine increased and that of aspartate decreased to the same extent on both sides following ammonia injection. After 7 days, the glutamate level decreased more in the intact than the decorticated side in both nonconvulsing and convulsing rats. The concentration of alanine increased most in the intact neostriatum, whereas glutamine levels increased and aspartate levels decreased to the same extent on both sides in nonconvulsing and convulsing rats. The results indicate that ammonia has a more pronounced effect on neuronal than glial glutamate pools.

Biochemical evidence for glutamate and/or aspartate as neurotransmitters in fibers from the visual cortex to the lateral posterior thalamic nucleus (pulvinar) in rats.

The effects of visual cortex ablation on several neurotransmitter parameters in the lateral thalamic nucleus (pulvinar) in rats have been investigated. We found a 57% decrease in high affinity uptake of D-[3H]aspartate in the pulvinar after ablation of the ipsilateral visual cortex. The KCl-evoked release of exogenous D-[3H]aspartate and endogenous glutamate were decreased by 33 and 37%, respectively. Moreover, the contents of endogenous glutamate and aspartate were decreased by 35%, each. The glutamate decarboxylase and choline acetyltransferase activities and the contents of other amino acids were not affected by the lesion. Our biochemical data indicate that glutamate and/or aspartate may be transmitters in the fibers from visual cortex to pulvinar in rats.

Effects of kainic acid and other excitotoxins in the rat superior colliculus: relations to glutamatergic afferents.

In this study we have performed surgical, chemical and combined surgical/chemical lesions in order to elucidate neurotransmitter mechanisms in the superior colliculus (SC) of albino rats. Visual cortex (VC) ablation reduced high affinity (HA) uptake of D-Asp by 32% in the deafferented SC. Local injection of kainic acid (KA) into SC reduced HA D-Asp uptake selectively in the lower dose range (less than 1 nmol) by 50-60%. The GABAergic marker glutamate decarboxylase (GAD) was decreased by maximally 60% only at doses exceeding 2 nmol. Choline acetyltransferase (ChAT), however, was not affected at any of the doses administered. VC ablation provided an almost complete protection against 1 nmol KA. When KA was injected 2 days prior to VC ablation an additive effect on HA D-Asp uptake of the two lesions was observed. From these observations we infer that the notion of a glutamatergic projection from VC to SC has been strengthened. Moreover, local neurons in intermediate layers account for about 60% of the HA D-Asp uptake in SC, and these are most likely impinged upon by the glutamatergic afferents. The neurotoxic effects of KA were compared with those of some suspected endogenous excitotoxins, i.e. N-methyl tetrahydrofolic acid (Me-THF), other folates and the tryptophan metabolite quinolinic acid (QA). N-methyl tetrahydrofolic acid, Me-THF (4 and 10 nmol) reduced HA D-Asp uptake by about 50%, only when coinjected with ascorbic acid. GAD and ChAT were not affected at either of the doses. QA was about 100-fold less potent than KA on a molar basis, and the maximal reduction of GAD was similar in QA and KA injected animals, whereas the maximal reduction of HA D-Asp was only 40% after QA injection in SC. We conclude that Me-THF, QA and KA exert their neurotoxic actions by different mechanisms as judged by the behavioral, histopathological and biochemical sequelae seen after local injections of the respective substances in intermediate layers of SC and corroborate data obtained from other brain areas.

A bioluminescence method for the measurement of L-glutamate: applications to the study of changes in the release of L-glutamate from lateral geniculate nucleus and superior colliculus after visual cortex ablation in rats.

We have developed a rapid, simple, specific, and very sensitive bioluminescence method for the measurement of L-glutamate (L-Glu). Oxidation of L-Glu by glutamate dehydrogenase has been coupled with bacterial FMN reductase and luciferase. Light production (i.e., peak height or integral) was linear from less than 0.5 to 500 pmol of L-Glu. Potential interfering substances that may be encountered in brain tissue have been identified. The most potent inhibitors were ascorbate and the biogenic amines. Procedures that conferred long-term stability of the reagent mixture (greater than 8 h) were established. Bioluminescence analysis of L-Glu content in brain tissue extracts, fractions from release experiments, and human CSF corroborated respective results obtained by HPLC analysis. In this study, we have applied the method to monitor changes in the KCl-evoked release of endogenous L-Glu from milligram amounts of brain tissue, i.e., from lateral geniculate nucleus and superior colliculus after visual cortex ablation.

Biochemical plasticity in the superior colliculus of adult rats after chronic visual cortex ablation.

We have measured the changes of several neurochemical parameters in the adult rat superior colliculus (SC) 1.5-4 months after unilateral visual cortex ablation. High-affinity uptake of D-[3H]Asp was increased by 22.5% and glutamate decarboxylase by 10% in the ipsilateral SC. This may be largely attributed to reactive synaptogenesis of intrinsic glutamatergic and GABAergic neurons, respectively. The noradrenaline content was increased by about 60 and 25% in the ipsi- and contralateral SCs, respectively. The latter probably reflects sprouting of noradrenergic fibres from the locus coeruleus.

Elevated concentrations of glutamate and aspartate in human ventricular cerebrospinal fluid (vCSF) during episodes of increased CSF pressure and clinical signs of impaired brain circulation.

In the ventricular cerebrospinal fluid (vCSF) of 10 hydrocephalic patients the mean (+/- S.D.) concentrations of glutamate and asparate were 2.9 +/- 0.2 and 0.2 +/- 0.2 microM, respectively. Significantly higher concentrations of these amino acids were found in two patients (glutamate 37.8 and 22.4 microM, aspartate 2.2 and 0.6 microM) with symptoms of impaired brain tissue perfusion, i.e. relative ischemia due to severely increased intraventricular CSF pressure. Our results are consistent with recent experiments in rats showing increased extracellular concentrations of glutamate and aspartate during transient cerebral ischemia.

Effects of area 17 ablation on neurotransmitter parameters in efferents to area 18, the lateral geniculate body, pulvinar and superior colliculus in the cat.

The result of unilateral ablation of visual cortical area 17 in adult cats was consistent with glutamate-aspartate being the neurotransmitter in efferents to the lateral geniculate body, the pulvinar and the visual part of superior colliculus but not in efferents to area 18 and the non-visual strata of superior colliculus. Furthermore, the distribution of glutamatergic, GABAergic and cholinergic markers within the various subdivisions of the cat visual system complied well with observations made previously with biochemical, neurophysiological, histochemical and immunohistochemical methods, in this and other mammalian species.

Development of neurotransmitter parameters in lateral geniculate body, superior colliculus and visual cortex of the albino rat.

The postnatal development of some neurotransmitter parameters was measured in lateral geniculate body, superior colliculus and visual cortex of the rat. The following parameters were studied: (i) high-affinity uptake of L-glutamate or D-aspartate as markers for glutamergic neurons; (ii) high-affinity uptake of GABA, which reflects both glial and neuronal uptake of GABA; (iii) HA beta-alanine uptake as a marker for accumulation of GABA in glial structures; (iv) activity of glutamic acid decarboxylase which reflects GABAergic neurons; and (v) activity of choline acetyltransferase as a cholinergic marker. Km and Vmax were determined for high-affinity uptake of glutamate and GABA in newborn and adult animals. The possible glial influence on the uptake during development is discussed. In lateral geniculate body and visual cortex the HA glutamate uptake showed increasing activity from birth to adulthood, whereas in superior colliculus, the uptake was higher at birth, reaching a small significant peak after 12 days of age, and was then reduced to adult level. Km showed no such change between neonatal and adult animals. At birth, high-affinity GABA-uptake was similar to the adult level in superior colliculus and lateral geniculate body. In visual cortex, the uptake of GABA was 50% of adults. However, on day 15, the GABA uptake showed 2 to 3-fold higher activity in all regions when compared to adult level. Km for GABA uptake in neonatals and adults differed only in lateral geniculate body. High affinity uptake of beta-alanine was 50-80% lower in adults than in newborn rats. Glutamate decarboxylase activity, however, increased continuously in all 3 regions examined. This was true also for choline acetyltransferase.

Polyamine-induced hydrolysis of apurinic sites in DNA and nucleosomes.

The ability of different polyamines to catalyze hydrolysis of phosphodiester linkages in apurinic and apyrimidinic (AP) sites has been investigated in supercoiled, relaxed and denatured DNA, and also in core and chromatosome particles. The rate constants for the hydrolysis in the DNAs have been determined. In general the order of effectiveness of the polyamines were: spermine greater than spermidine greater than putrescine greater than cadaverine. A 9 fold difference in rate constants was found between spermine and cadaverine. No difference in the rate of hydrolysis was seen between AP-sites in supercoiled and relaxed DNAs, whereas the rate for the single-stranded DNA and DNA in core and chromatosome particles was only half of that in the double-stranded DNA. All AP-sites in both free DNA and DNA-histone particles were hydrolyzed in the presence of polyamines. For all polyamines, with the exception of spermine, increasing concentration of both Mg++ and salts such as KCl both led to a large decrease in the rate of polyamine-induced hydrolysis of AP-sites. The rate of hydrolysis increased markedly with increasing pH in the pH range pH 6 - pH 11.