Brain Amino Acids Measured by Intracerebral Dialysis in Portacaval Shunted Rats

Changes in brain amino acid uptake and metabolism have been proposed as a possible etiological factor in hepatic encephalopathy. By use of a brain dialysis technique (a thin tube implanted in the brain of the living animal), the extracellular amino acid concentrations in the striatum of portacaval (PC)-shunted and sham-operated rats were measured. Leucine, phenylalanine, methionine, and glutamine were increased two- to sixfold in the PC-shunted rats, whilst no changes were seen for GABA, valine, glutamate, or isoleucine, confirming previous reports. Aspartate levels were 350% higher in the PC-shunted rats, and this rise, as well as that of phenylalanine, was significantly correlated with the lower motor activity observed in the PC-shunted rats, suggesting a possible importance of these amino acids in the etiology of hepatic encephalopathy. The amino acid concentrations measured in whole blood demonstrated the well-known pattern of low levels of branched-chain amino acids and increased concentrations of phenylalanine, glutamine, and histidine.     

Functional Activity of Substantia Nigra Grafts Reinnervating the Striatum: Neurotransmitter Metabolism and [14C]2-Deoxy-d-glucose Autoradiography

Dopaminergic innervation of the caudate nucleus in adult rats can be partially restored by the grafting of embryonic substantia nigra into the overlying parietal cortex with concomitant compensation of certain behavioral abnormalities. In this study the function of such grafts was investigated neurochemically by quantification of transmitter metabolism and glucose utilization in the reinnervated target. Rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal bundle received a single graft to the dorsal caudate-putamen and were screened for rotational behavior following 5 mg/kg methamphetamine. The grafts restored dopamine concentrations in the caudate-putamen from initially less than 0.5% to an average of 13.6% of normal in rats with behavioral compensation. The ratio of 3,4-dihydroxyphenylacetic acid to dopamine, which is a measure of the rate of transmitter turnover, were equivalent in transplanted and normal control rats. Moreover, measurements of DOPA accumulation for a 30-min period after DOPA decarboxylase inhibition indicated similar fractional dopamine turnover rates in normal and transplant-reinnervated tissues. Correlations between rotational behavior and dopamine concentrations showed that reinnervation to only 3% of normal was sufficient to counterbalance the motor asymmetry. Measurements of glucose utilization by [14C]deoxyglucose autoradiography indicated equivalent metabolic rates for the grafted tissue and the intact substantia nigra. 6-Hydroxydopamine denervation of the caudate-putamen had no significant effect on neuronal metabolism in that region, nor did subsequent reinnervation from a graft. Grafts, however, were associated with a 16% reduction of glucose uptake in the ipsilateral globus pallidus, indicating a significant transsynaptic influence of the nigral transplants on neuronal metabolism in the host brain. Overall the results indicate that behaviorally functional neuronal grafts spontaneously metabolize dopamine and utilize glucose at rates characteristic of the intact nigrostriatal system. This provides further evidence that ectopic intracortical nigral transplants can reinstate dopaminergic neurotransmission in regions of the host brain initially denervated by the 6-hydroxydopamine lesion.     

Inhibition of the conversion of glycine to serine in spinach leaf mitochondria

Isonicotinyl hydrazide, glycine hydroxamate, aminoacetonitrile and KCN inhibited the conversion of glycine to serine in spinach ( Spinacea oleracea L. cv. Viking II) mitochondria. The site of inhibition for the different inhibitors was studied. Isonicotinyl hydrazide and glycine hydroxamate both inhibited the partial reactions glycine-bicarbonate exchange and serine hydroxymethyltransferase. The inhibition was competitive for the exchange reaction and noncompetitive for serine hydroxymethyltransferase. Aminoacetonitrile at low concentration (1 m M ) inhibited the glycine-bicarbonate exchange specifically, whereas serine hydroxymethyltransferase was inhibited only at higher concentrations. Aminoacetonitrile was a competitive inhibitor for both reactions. The serine hydroxymethyltransferase was inhibited by KCN whereas the glycine-bicarbonate exchange was only partially inhibited. The KCN-inhibition of serine hydroxymethyltransferase was competitive.     

Effects of bud removal in Scots pine (Pinus silvestris) seedlings

One- and two-“year”-old seedlings of Pinus silvestris L., from which the buds had been removed, were studied for five weeks during the second and third growth period, respectively. Intact seedlings were used as controls. The seedlings were cultivated under controlled conditions in a climate chamber. The growth of the seedlings was determined and the one-“year”-old needles assayed for changes in net photosynthesis and ribulose bisphosphate carboxylase activity and in the levels of protein, Kjeldahl nitrogen, chlorophyll and starch. In the control the carboxylase activity and the content of protein, Kjeldahl nitrogen and starch in the needles increased in the beginning of the “summer” and decreased during the shoot growth period. The starch content was higher after bud removal (decapitation), since the carbohydrate could not be utilized for the growth of the new shoot. Decapitation did not affect the growth rate of the roots. The content of Kjeldahl nitrogen and total and soluble protein in the needles was higher in the decapitated seedlings during the period of shoot elongation in the control. Total nitrogen, but not protein, reached high levels, indicating accumulation of non-protein compounds. The general course of the chlorophyll pattern was not affected. Higher ribulose bisphosphate carboxylase activity than in the control was observed in the later part of the experimental periods. The higher levels of protein and nitrogen as well as of carboxylase activity after decapitation support the interpretation that soluble protein, including the carboxylase, and possibly other nitrogen compounds in the older needles are used for growth of the shoot. The loss of protein and nitrogen and of carboxylase activity in the control did not seem to be due to mineral deficiency in the substrate. Despite higher levels of carboxylase activity and similar chlorophyll concentrations, light-saturated net photosynthesis was lower after decapitation. The ratio between photosynthesis and photorespiration was not affected.     

Telomere-associated repeats in Chironomus form discrete subfamilies generated by gene conversion

Summary In dipteran insects the most distal telomere-associated DNA known to exist consists of long, complex tandem repeats. We have classified the 340-bp tandemly arranged repeats in Chironomus pallidivittatus. The repeats are distributed in a small number of subfamilies. One type of the repeat has the character of a master unit from which other main units can be derived usually by simple changes. The derived subfamilies contain segments that are degenerate versions of the corresponding segment in the master sequence. Such segments can also occur together in one and the same repeat unit in different combinations. There is a complete absence of subfamily-specific base variants in regions lying outside of the degenerate segments. Homogenization takes place between DNA sequences that are often smaller than a whole repeat unit. The mosaic structure of the repeat arrays suggests that gene conversion is an important force in the generation and maintenance of this family of repeats.Offprint requests to: M. Cohn     

In Vivo Activation of Kainate Receptors Induces Dephosphorylation of the Heavy Neurofilament Subunit

Injection of kainic acid (KA) into the rat hippocampus reduced the phosphorylation-related immunoreactivity of the heavy subunit of neurofilament proteins (NF-H). The effect was demonstrated quantitatively with a dot-immunobinding assay and qualitatively by immunoblotting with monoclonal antibodies against phosphorylation-dependent and nonphosphorylation-related epitopes of NF-H. The KA-induced reduction affected 50% of the phosphorylated NF-H in half of the hippocampus after 48 h. At the same time, the nonphosphorylation-related NF-H immunoreactivity increased as revealed by immunoblotting, indicating a shift from phosphorylated to nonphosphorylated NF-H. The effects on NF-H preceded a decrease in content of the neuron-specific enolase, a soluble neuronal cytoplasmic protein. No alterations of the light subunit of neurofilament proteins occurred, suggesting that KA has a preferential effect on NF-H phosphorylation. N-Methyl-D-aspartate administered similarly did not lead to a rapid dephosphorylation of NF-H. We propose that kainate receptor-mediated dephosphorylation in NF-H is involved in the signal transduction of excitatory amino acids with consequences for neuronal functions dependent on intermediary filament phosphorylation.     

Mobility restriction in vivo of the heavy ribosomal subunit in a secretory cell

Analysis in insect (Chironomus tentans) salivary gland cells of labeled RNA as a function of time after precursor injection and its distance to the nuclear membrane, cytoplasmic zone analysis, could previously be used to demonstrate the presence of short-lasting gradients in newly labeled ribosomal RNA. Since the gradients were sensitive to puromycin, they are likely to be a result of diffusion restriction due to an engagement of the subunits into polysomes. In the present paper the possibility was explored of recording gradients that were caused by labeled subunits in puromycin-resistant associations, which, in all probability, involve the endoplasmic reticulum. It was found that labeled 28 S and 5 S RNA but not 18 S RNA were present in radioactivity gradients lasting for at least 2 days but less than 6 days. The gradients also remained during inhibition of RNA synthesis by actinomycin, and they were completely resistant to puromycin whether given in vivo or in vitro. The semipermanent gradients formed fhere offer a unique parameter for the in vivo study of conditions for formation and maintenance of heavy subunits in puromycin-resistant bonds. An explanation for these and previous results is that the light subunit, although restricted in movement by engagement to polysomes, is nevertheless free to exchange and spread between rounds of translation, whereas at least part of the heavy subunit population is bound to the endoplasmic reticulum and less free to spread. These results offer a good in vivo correlate to previous results on in vitro exchangeability of subunits.     

Balbiani ring RNA content and half-life in nucleus and cytoplasm of Chironomus tentans salivary gland cells

The content of RNA with an origin in the Balbiani rings 1 and 2 (BR 1+2) has been determined in chromosomes, nuclear sap and cytoplasm of Chironomus tentans salivary gland cells. Together with information on rate and completeness of export this permits an estimation of half-life of this RNA in cytoplasm and its residence time in the nucleus. The quantities in the BR, nuclear sap, and cytoplasm are roughly related as 110200. The 75 S RNA in the nuclear sap with an origin in the BR 1+2 must to a high extent be a precursor to the cytoplasmic 75 S RNA in vivo. The half-life of the cytoplasmic component is about 20 h and the half-life (residence time) for BR 1+2 RNA in the nuclear sap around one hour. The presence of a large pool of BR RNA in the sap explains the previously observed delay in its cytoplasmic appearance in vivo.     

Physico-chemical properties of chromosomal RNA in Chironomus tentans polytene chromosomes

Labelled chromosomal RNA of the dipteran Chironomus tentans was studied with respect to its migration properties during electrophoresis in agarose. The RNA was isolated from polytene chromosomes which had been microdissected from fixed salivary glands and obtained free from nucleoli and nuclear sap. Labelled material migrates as 4–5 S RNA and as polydisperse material in a range where the lower limit corresponds to 10–15 S, the upper limit to 80–90 S RNA and the maximum in the distribution to 30–40 S RNA. The data indicate that the latter fractions are formed by unbroken, single-stranded RNA molecules, partly of very high molecular weights. It is shown in a number of tests that the distribution is not a consequence of formation of complexes or aggregates between RNA molecules on one hand and DNA, proteins or other RNA molecules on the other.