The effect of Cd2+ on the biosynthesis of chlorophyll in leaves of barley

The effect of cadmium on the biosynthesis of chlorophyll has been investigated in the leaves of dark-grown seedlings of barley ( Hordeum vulture L. cv. Proctor). Cd2+ inhibited the production of chlorophyll by affecting 1) the synthesis of 5-aminolacvulinic acid and 2) the protoehlorophyllide reductase ternary complex with its substrates. Cd2+ had no effect on the constituent enzymes that catalyse the synthesis of free protoehlorophyllide from 5-aminolaevulinic acid. The results obtained are consistent with Cd2+ inhibiting the formation of chlorophyll by reacting with essential thiol groups in both the protochlorophyllide reductase protein and the enzyme(s) involved in the light dependent synthesis of 5-aminolaevulinic acid.     

Inhibition of geranylgeranyl diphosphate synthesis in in vitro systems

The incorporation of [¹⁴C]mevalonate and [¹⁴C]isopentenyl diphosphate into geranylgeranyl diphosphate was investigated in in vitro systems from Cucurbita pepo (pumpkin) endosperm and from Avena sativa etioplasts. Mevalonate incorporation was effectively inhibited in the pumpkin system by geranylgeranyl diphosphate and geranylgeranyl monophosphate but less effectively by phytyl diphosphate or inorganic diphosphate. Membrane lipids, geranyllinalool, or lecithin enhanced mevalonate incorporation in the Cucurbita system. Incorporation of isopentenyl diphosphate was also enhanced by lecithin and inhibited by geranylgeranyl diphosphate in the Cucurbita system. No lipid enhancement was found in the Avena system; inhibition by GGPP required a much higher GGPP concentration than in the Cucurbita system.     

Diterpenoids from Caesalpinia pulcherrima

Three new furanoditerpenoids of the caesalpin-type have been isolated from the roots of Caesalpinia pulcherrima. The structures of these compounds, vouacapen-5α-ol, 6β-cinnamoyl-7β-hydroxy-vouacapen-5α-ol and 8,9,11,14-didehydrovouacapen-5α-ol, were elucidated through interpretation of their spectral data. Sitosterol was also obtained.     

Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L.

5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase; EC 2.5.1.19), 3-dehydroquinate dehydratase (EC 4.2.1.10) and shikimate: NADP⁺ oxidoreductase (EC 1.1.1.25) were present in intact chloroplasts and root plastids isolated from pea seedling extracts by sucrose and modified-silica density gradient centrifugation. In young (approx. 10-d-old) seedling shoots the enzymes were predominantly chloroplastic; high-performance anion-exchange chromatography resolved minor isoenzymic activities not observed in density-gradientpurified chloroplasts. The initial enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was also associated with intact density-gradient-purified chloroplasts. 3-Dehydroquinate synthase (EC 4.6.1.3) and shikimate kinase (EC 2.7.1.71) were detected together with the other pathway enzymes in stromal preparations from washed chloroplasts. Plastidic EPSP synthase was inhibited by micromolar concentrations of the herbicide glyphosate.Abbreviations DAHP 3-deoxy-d-arabino-heptulosonate 7-phosphate - DEAE diethylaminoethyl - DHQase 3-dehydroquinate dehydratase - DTT dithiothreitol - EPSP 5-enolpyruvylshikimate 3-phosphate - SORase shikimate:NADP⁺ oxidoreductase     

Effects of p,p''-DDT on the Rat Brain Concentrations of Biogenic Amine and Amino Acid Neurotransmitters and Their Association with p,p''-DDT-Induced Tremor and Hyperthermia

Male, Fischer strain 344 adult rats were given various doses (25-100 mg/kg) of p,p'-DDT by oral gavage, and levels of biogenic amines, their metabolites, and amino acid neurotransmitters, tremor activity, and rectal temperature were measured at several intervals (2, 5, 12, and 24 h) after dosing. Dose-related increases in rectal temperature and in tremor activity were observed at 50-100 mg/kg 12 h after dosing. Tremorigenic doses of DDT increased the 5-hydroxyindoleacetic acid (5-HIAA) level in hypothalamus, brainstem, and striatum, whereas doses of 75 and 100 mg/kg increased the 3-methoxy-4-hydroxyphenylglycol (MHPG) level in hypothalamus and brainstem and the 3,4-dihydroxyphenylacetic acid level in striatum. Six amino acids were assayed in the brainstem, hypothalamus, and striatum; aspartate and glutamate levels were increased only in brainstem at 25-100 mg/kg. No consistent changes in concentrations of taurine, glutamine, glycine, or gamma-aminobutyric acid were observed in any of the regions assayed. Time-related increases in rectal temperature were seen 2-12 h after dosing, and the presence of tremor was observed 5-12 h after dosing; for both the time of peak effect was at 12 h. The DDT-induced hyperthermia and tremor were associated with dose- and time-related increases in levels of 5-HIAA, MHPG, aspartate, and glutamate. It is suggested that an increase in the turnover rate of 5-hydroxytryptamine (5-HT) may be responsible for the DDT-induced hyperthermia, whereas increases in the metabolism of 5-HT and norepinephrine may be involved in the tremor.     

Influence of δ-Opioid Receptors on Production of Labeled Methionine5-Enkephalin in Murine Neuroblastoma Cells

Synthesis of methionine5-enkephalin by intact cells of murine neuroblastoma clone N1E-115 has been demonstrated both immunocytochemically and biochemically. In addition, N1E-115 cells possess homogeneous enkephalin (delta) receptors which inhibit prostaglandin E1-induced intracellular cyclic AMP formation. An assay was developed for measuring de novo synthesis of methionine5-enkephalin by pulsing cells in culture with radioactive methionine and isolating this pentapeptide to radiochemical purity by a procedure that included immunoaffinity chromatography specific for oxidized methionine5-enkephalin. This assay indicated that production of radiolabeled-methionine5-enkephalin was increased upon lengthy exposure of intact N1E-115 cells in the late logarithmic phase of growth to a nonproteolyzable analog of methionine5-enkephalin. This increase in synthesis of intracellular methionine5-enkephalin relative to control cells was prevented by prior incubation of the clone with naloxone, indicating that the response was mediated by the delta receptor.     

Antisera Against the Indolealkylamines: Tryptophan, 5-Hydroxytryptophan, 5-Hydroxytryptamine, 5-Methoxytryptophan, and 5-Methoxytryptamine Tested by an Enzyme-Linked Immunosorbent Assay Method

Antisera were raised against tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, 5-methoxytryptophan, and 5-methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme-linked immunosorbent assay method. The specificity of each anti-indolealkylamine-glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly-L-lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross-reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.     

Evidence for [D-Ala2,D-Leu5]Enkephalin-Induced Supersensitivity to 5-Hydroxytryptamine in a Neurotumor × Brain Hybrid Cell Line (NCB-20)

A neuroblastoma X Chinese hamster embryonic brain explant hybrid cell line (NCB-20) expressed 5-hydroxytryptamine (5-HT1) receptors, linked to adenylate cyclase, which closely resembled 5-HT1 receptors previously characterized in central nervous tissue. However, the affinity of the receptors for 5-HT was only 150 nM compared to 5 nM in membranes prepared from cerebral cortex. The elevation of cyclic AMP levels in NCB-20 cells produced by 5-HT was found additive to that produced by cholera toxin but synergistic with that produced by either prostaglandin E1 (PGE1) or forskolin, suggesting that these latter two agents elevate cyclic AMP levels by a different mechanism than 5-HT. The elevation of cyclic AMP levels by either 5-HT or PGE1 was reversed by [D-Ala2,D-Leu5]enkephalin (DADLE), morphine, clonidine, and 3,4-dihydroxyphenylethylamine (dopamine) on a short (30 min) time scale. However, continued exposure to DADLE resulted in loss of the initial inhibitory effects of DADLE after 6 h and return of cyclic AMP levels to that seen with either 5-HT or PGE1 alone. When the DADLE exposure time was increased to 48 h, 5-HT produced a further twofold increase in cyclic AMP levels, but there was no increase in the responsiveness of the cells to PGE1 unless naloxone was added 1 h prior to treatment with PGE1. Scatchard analysis showed that the increased potency of 5-HT resulted from an increase in receptor affinity for 5-HT (from a KD of 150 +/- 20 nM to one of 20 +/- 7 nM), with a reduction in the number of apparent binding sites. The 5-HT supersensitivity observed in NCB-20 cells may be a good model for neurotransmitter interactions that produce desensitization or facilitation in the intact nervous system.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

The biological relevance of HPLC-purified vasoactive intestinal polypeptide monoiodinated at tyrosine 10 or tyrosine 22

Three major forms of monoiodinated VIP (M125I-VIP) were isolated after chloramine-T iodination and HPLC purification. The iodinated tyrosine residue was located in each form of M125I-VIP using arginase C and trypsin digestion for obtaining defined fragments containing only one tyrosine residue. The HPLC isolated iodinated fragments thus obtained were used for HPLC comigration studies with iodinated synthetic C and N terminal VIP fragments and for amino acid analysis. The first two eluting peaks 1 and 2 are (M125I-Tyr10-VIP); peak 1 has an oxidized methionine; peak 3 is a (M125I-Tyr22-VIP) which also has an oxidized methionine. A reduced counterpart of peak 3 named peak 4 was isolated by further HPLC analysis. The ability of the different species of M125I-VIP to stimulate adenosine cyclic 3',5'-phosphate (cAMP) production in transformed colonic cells in culture (HT-29) was compared to that of native VIP. The mean potencies of the M125I-VIP species expressed as a percentage relative to the potency of native VIP were, peak (1): 0.98; (2): 0.84; (3): 1.38; (4): 1.48, in the range of concentrations tested (2-60 pM). The M125I-Tyr22-VIP are significantly more active than native VIP (P less than 0.01). Oxidation of methionine or iodination of tyrosine 10 does not significantly modify the biological activity of VIP. We conclude that iodination of Tyr-22 located in the apolar helical COOH-terminal of VIP increases the effectiveness of VIP interaction with its receptors. Thus the tyrosyl residue and the localized hydrophobic features of VIP are critically involved in the function of this neurotransmitter.