Light-independent accumulation of chlorophyll a and b and protochlorophyllide in green barley (Hordeum vulgare)

Barley ( Hordeum vulgare L. cvs Clipper, Procter, Astrix) seedlings were transferred from daylight to darkness and changes in chlorophyll a , chlorophyll b , protochlorophyllide and chlorophyllide (μ leaf⁻¹) in either the first or second leaf determined spectrophotometrically after separating the esterified from unesterified pigments by partitioning between ammoniacal acetone and light petroleum ether. Chlorophyll a and b as well as protochlorophyllide accumulated in the dark. The ratio of chlorophyll to protochlorophyllide formed in the absence of light was 18:1. 5-aminolevulinic acid (10 m M ) promoted the synthesis of chlorophyll a and b and protochlorophyllide. Pigment synthesis and response to 5-aminolevulinic acid addition was related to tissue age. Mature tissue in the apical third of the leaf accumulated most chlorophyll, but per μg chlorophyll present at the time of transfer to darkness, was less efficient than immature tissue towards the base of the leaf. Immature tissue was also most responsive to added 5-aminolevulinic acid. Chlorophyll synthesis in the dark was accompanied by chloroplast development. Chloroplasts in immature leaf tissue increased in size and extent of thylakoid development when transferred from daylight to darkness. The results indicate that chlorophyll synthesis and chloroplast membrane development in light-grown barley continue into the dark phase of the diurnal cycle. A light-independent protochlorophyllide reductase in light-grown barley seedlings is postulated.     

Potential bioactivation pathways for the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

The metabolism of the selective nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been studied in rat brain mitochondrial incubation mixtures. The 1-methyl-4-phenylpyridinium species MPP+ has been characterized by chemical ionization mass spectral and 1H NMR analysis. Evidence also was obtained for the formation of an intermediate product which, with the aid of deuterium incorporation studies, was tentatively identified as the alpha-carbon oxidation product, the 1-methyl-4-phenyl-2,3-dihydropyridinium species MPDP+. Comparison of the diode array UV spectrum of this metabolite with that of the synthetic perchlorate salt of MPDP+ confirmed this assignment. The oxidation of MPTP to MPDP+ but not of MPDP+ to MPP+ is completely inhibited by 10(-7) M pargyline. MPDP+, on the other hand, is unstable and rapidly undergoes disproportionation to MPTP and MPP+. Based on these results, we speculate that the neurotoxicity of MPTP is mediated by its intraneuronal oxidation to MPDP+, a reaction which appears to be catalyzed by MAO. The interactions of MPDP+ and/or MPP+ with dopamine, a readily oxidizable compound present in high concentration in the nigrostriatum, to form neurotoxic species may account for the selective toxic properties of the parent drug.     

Effect of sodium sulfate on in vitro organogenesis of tobacco callus

Callus of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) was grown on callus-proliferating (CP) and shoot-forming (SF) media with elevated sodium sulfate (Na₂SO₄) concentrations either in the light or dark for more than one year. An increase in Na₂SO₄ concentration resulted in a decrease in callus growth index, an increase in percent dry weight of callus tissues grown on both media, and a decrease in both number of calli forming shoots and number of shoots per callus in SF medium. The CP callus grown in the light spontaneously began to form shoots after the 5th monthly transfer, and spontaneous root formation occured after the 16th transfer in the presence of 0.75 and 1.0% Na₂SO₄. Both water () and osmotic (_s) potentials of the callus increased with increasing Na₂SO₄ concentration; and callus exhibited greater and _s in the light than dark for both CP and SF media.     

Active uptake of MPP+, a metabolite of MPTP, by brain synaptosomes

Mouse brain synaptosomal preparations were used to study uptake of N-methyl-4-phenylpyridine (MPP+), a metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). The uptake of [3H]-MPP+ by striatal synaptosomes was approximately 25 X greater than that of [3H]-MPTP, with a KM of 0.48 microM and a Vmax of 5.3 nmoles/g tissue/min. Uptake was Na+ dependent and inhibited by ouabain, cocaine and dopamine (Ki 0.12 microM). Synaptosomes prepared from the corpus striatum accumulated [3H]-MPP+ at a rate 5-10 times higher than preparations from other brain regions. This selective uptake of MPP+ may contribute to the specificity of the toxic effects of MPTP on nigrostriatal dopaminergic neurons.     

Oestrogen-induced cholesterol and fatty acid biosynthesis in Xenopus laevis liver during vitellogenic response

1. Oestradiol-17β induces livers of Xenopus laevis (South African clawed toad) to synthesize and secrete into the serum large quantities of the egg-yolk-protein precursor, vitellogenin. The peak of this response occurs 9–16 days after hormone treatment [Dolphin, Ansari, Lazier, Munday & Akhtar (1971) Biochem. J. 124, 751–758]. It is now shown that 6 days after hormone treatment a 120–160-fold stimulation of the synthesis of cholesterol and fatty acid compared with control values occurred. 2. A cell-free system, derived from Xenopus liver, which synthesizes squalene and fatty acid is described. By using this system, several hundredfold stimulation of incorporation of [¹⁴C]acetate into squalene was recorded 6 days after the administration of oestradiol-17β, compared with a 3–4-fold stimulation of incorporation of [³H]mevalonate compared with control values. It is argued that oestradiol-17β must affect enzyme(s) catalysing step(s) between acetate and mevalonate in the biosynthetic pathway to cholesterol. 3. In incubation of liver slices in vitro, most of the lipid and cholesterol synthesized in response to the steroid hormone was associated with those subcellular fractions that contained membranes. Moreover, pulse-labelling experiments in vivo showed that 70% of this lipid and cholesterol was retained in the liver. The remainder appeared in the serum, where it was equally distributed between vitellogenin and vitellogenin-free serum. 4. G.l.c. analyses of the cholesterol content of liver microsomal fractions of Xenopus laevis indicated that the cholesterol content was at least 50% higher in microsomal fractions obtained from livers that had been exposed to oestradiol-17β. Meanwhile, g.l.c. analysis of the lipid moiety of secreted vitellogenin showed that up to 35% of its lipid was cholesterol.     

Changes in isoperoxidases during shoot formation in tobacco callus

Summary Shoot formation in tobacco (Nicotiana tabacum L.) callus is accompanied by an increase in peroxidase activity which takes a form similar to a sigmoid curve. The “stationary” phase coincide with the period of organ formation. Characteristic changes in isoperoxidase pattern are found in the shoot-forming part of the callus. These changes are different from those in the nonshoot-forming part or in gibberellin-treated tissue, which does not form shoots.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Mature DIABLO/Smac is produced by the IMP protease complex on the mitochondrial inner membrane

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX(5)P in Imp1 and NX(5)S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space.     

Towards building the tree of life: a simulation study for all angiosperm genera

Comprehensive phylogenetic trees are essential tools to better understand evolutionary processes. For many groups of organisms or projects aiming to build the Tree of Life, comprehensive phylogenetic analysis implies sampling hundreds to thousands of taxa. For the tree of all life this task rises to a highly conservative 13 million. Here, we assessed the performances of methods to reconstruct large trees using Monte Carlo simulations with parameters inferred from four large angiosperm DNA matrices, containing between 141 and 567 taxa. For each data set, parameters of the HKY85+G model were estimated and used to simulate 20 new matrices for sequence lengths from 100 to 10,000 base pairs. Maximum parsimony and neighbor joining were used to analyze each simulated matrix. In our simulations, accuracy was measured by counting the number of nodes in the model tree that were correctly inferred. The accuracy of the two methods increased very quickly with the addition of characters before reaching a plateau around 1000 nucleotides for any sizes of trees simulated. An increase in the number of taxa from 141 to 567 did not significantly decrease the accuracy of the methods used, despite the increase in the complexity of tree space. Moreover, the distribution of branch lengths rather than the rate of evolution was found to be the most important factor for accurately inferring these large trees. Finally, a tree containing 13,000 taxa was created to represent a hypothetical tree of all angiosperm genera and the efficiency of phylogenetic reconstructions was tested with simulated matrices containing an increasing number of nucleotides up to a maximum of 30,000. Even with such a large tree, our simulations suggested that simple heuristic searches were able to infer up to 80% of the nodes correctly.