Effects of vanadate, N2 and light on the membrane potential of motor cells and the lateral leaflet movements of Desmodium motorium

The lateral leaflets of Desmodium motorium (Houtt.) Merr. exhibit ultradian up- and down movements, which are paralleled by oscillations of the membrane potential of motor cells in the pulvinus. By different treatments we have tested the hypothesis that both that both oscillation-types are causally related. The reactions of the leaflet movement and the membrane potential were evaluated by the following approaches. (1) Application of vanadate. an inhibitor of the proton pump in the plasmalemma. and N₂ suppressed leaflet movements and finally arrested the leaflet in the lower position. Before the oscillations damped out, a strong lengthening in period was found. This indicates that the pump is part of the ultradian clock. A period lenthening and a final suppression of the rhythm by vanadate was also seen in the extracellular electric potential of the pulvinus. Intracellular recordings in situ showed that vanadate application depolarized the motor cells. (2) Light of high fluence rates diminished the amplitude of the oscillations of the membrane potential of single motor cells and shortened the period. The same effects were observed when monitoring the lateral leaflet movement. The leaflet always moved towards the direction of the light. whether it was applied from the abaxial or from the adaxial part of the pulvinus. (3) When light was applied to the pulvinus of lateral leaflets. which had spontancously stopped moving in an upper position. oscillations were induced transiently. This effect was also found for the membrane potential of motor cells in the pulvinus. - Our results thus provide further evidence that the membrane potential controls the volume state of the motor cells in the pulvinus of lateral leaflets of Desmodium motorium .     

Production of the new antimalarial drug artemisinin in shoot cultures of Artemisia annua L.

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l^-1 BAP, 0.05 mg l^-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l^-1 gibberellic acid, 0.5 g l^-1 casein hydrolysate, 10 mg l^-1 or 20 mg l^-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.Abbreviations BAP benzylaminopurine - DW dry weight - FW fresh weight - GA₃ gibberellic acid - MS Murashige & Skoog basal medium - NAA naphthaleneacetic acid     

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.     

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.     

Complete characterization of a large marker chromosome by reverse and forward chromosome painting

Marker chromosome are small supernumerary chromosomes that are sometimes associated with developmental abnormalities. Hence, the genes involved in such cases provide an interesting approach to understanding developmental abnormalities in man. As a first step towards isolating such sequences, marker chromosomes need complete characterization. By combining chromosome isolation by flow sorting and the degenerate oligonucleotide primed — polymerase chain reaction, we have constructed a DNA library specific for a marker chromosome found in a child with severe developmental abnormalities. We used fluorescent in situ hybridization of the library onto normal metaphase spreads (reverse chromosome painting) and were thus able to determine that the marker consists of the centromeric part of chromosome 7, the telomeric region of the long arm of chromosome 5 and the telomeric region of the short arm of the X-chromosome. Subsequently, we hybridized normal chromosome-specific libraries of the relevant chromosomes onto metaphases containing the marker chromosome (forward chromosome painting) and could in this manner establish the precise location of the different chromosome regions on the marker chromosome itself. This is a general approach suitable for outlining marker chromosomes in detail, and will aid the identification of the genes involved.