RNA polymerisation capacity and permeability to ribonucleoside triphosphates of nuclei from livers of wholebody X-irradiated rats

Summary For 4–18 h following whole-body X-irradiation of rats, liver nuclei showed a progressive increase in the permeability to ribonucleoside triphosphates (as assessed in vitro using tritiated uridine triphosphate (UTP)) and in the capacity to polymerise RNA in vitro (Mg⁺⁺-containing and Mn⁺⁺/(NH₄)₂SO₄-containing assay systems).     

Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of α-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes. Received 30 December 1998; accepted 12 January 1999     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> possesses a unique dual-specificity serine/threonine and tyrosine kinase,Pfnek3

Despite the absence of classical tyrosine kinases encrypted in the kinome of Plasmodium falciparum, biochemical analyses have detected significant tyrosine phosphorylation in its cell lysates. Supporting such phosphorylation is critical for parasite development. These observations have thus raised queries regarding the plasmodial enzymes accountable for tyrosine kinase activities in vivo. In the current investigation, immunoblot analysis intriguingly demonstrated that Pfnek3, a plasmodial mitogen-activated protein kinase kinase (MAPKK), displayed both serine/threonine and tyrosine kinase activities in autophosphorylation reactions as well as in phosphorylation of the exogenous myelin basic protein substrate. The results obtained strongly support Pfnek3 as a novel dual-specificity kinase of the malarial parasite, even though it displays a HGDLKSTN motif in the catalytic loop that resembles the consensus HRDLKxxN signature found in the serine/threonine kinases. Notably, its serine/threonine and tyrosine kinase activities were found to be distinctly influenced by Mg²⁺ and Mn²⁺ cofactors. Further probing into the regulatory mechanism of Pfnek3 also revealed tyrosine phosphorylation to be a crucial factor that stimulates its kinase activity. Through biocomputational analyses and functional assays, tyrosine residues Y117, Y122, Y172, and Y238 were proposed as phosphorylation sites essential for mediating the catalytic activities of Pfnek3. The discovery of Pfnek3’s dual role in phosphorylation marks its importance in closing the loop for cellular regulation in P. falciparum, which remains elusive to date.     

The antifertility action ofa-chlorohydrin: Enzyme inhibition bya-chlorohydrin phosphate

Summary Preparations of the enzymes glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase are shown to be inhibited bya-chlorohydrin phosphate (II) in a competitive and non-competitive manner, respectively.a-Chlorohydrin (I), glycidol and epi-chlorohydrin have no inhibitory activities suggesting that their antifertility actions are due to their metabolism in vivo toa-chlorohydrin phosphate.     

Targeting O 6-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy

O ⁶-methylguanine-DNA methyltransferase (MGMT) repairs the cancer chemotherapy-relevant DNA adducts, O ⁶-methylguanine and O ⁶-chloroethylguanine, induced by methylating and chloroethylating anticancer drugs, respectively. These adducts are cytotoxic, and given the overwhelming evidence that MGMT is a key factor in resistance, strategies for inactivating MGMT have been pursued. A number of drugs have been shown to inactivate MGMT in cells, human tumour models and cancer patients, and O ⁶-benzylguanine and O ⁶-[4-bromothenyl]guanine have been used in clinical trials. While these agents show no side effects per se, they also inactivate MGMT in normal tissues and hence exacerbate the toxic side effects of the alkylating drugs, requiring dose reduction. This might explain why, in any of the reported trials, the outcome has not been improved by their inclusion. It is, however, anticipated that, with the availability of tumour targeting strategies and hematopoetic stem cell protection, MGMT inactivators hold promise for enhancing the effectiveness of alkylating agent chemotherapy.     

The cephalopod heart: The evolution of a high-performance invertebrate pump

Cephalopods typically have high metabolic rates. They have blood in which the oxygen carrier is haemocyanin, a pigment that is found only in solution and which never seems to be present in concentrations that will transport more than 4–5 vols % of oxygen. Their hearts must in consequence have very high cardiac outputs. In this account the performance of the heart ofNautilus, the only surviving ectocochleate, is contrasted with the performance of the hearts of coleoids,Octopus which has a relatively low metabolic rate (for a coleoid) and squids which have very high oxygen uptakes by any standards. In all these animals, heartbeat frequency is temperature-dependent and the additional oxygen demand in exercise is met very largely by a 2–3-fold increase in stroke volume. With the exception ofNautilus, cephalopods tend to utilise nearly all of the oxygen transported in the blood even at rest; they show very limited factorial scopes. Specific power output has, however, increased dramatically from 2.7 mWg^–1 in an activeNautilus to 5.5 mWg^–1 inOctopus and up to 20 or 30 mWg^–1 in species ofLoligo. The increase is almost entirely due to a 10-fold increase in heartbeat frequency. It is argued that frequency cannot be used as a means of responding to extra demand in an animal that must also carry automatic compensation for changes in metabolic rate dependent upon the ambient temperature, and that the use of frequency in some squid may be associated with a reduced temperature tolerance. Cephalopod systemic hearts do not scale directly with body mass, like the hearts of fish and the higher vertebrates. Smaller cephalopods have relatively larger hearts (as Mass^0.9). A typical 100-g coleoid would have a heart mass of 0.15 g. Oegopsid squids appear to be exceptional with hearts twice as large.     

Serotonin-stimulated protein phosphorylation in aortic smooth muscle cells

Summary The effects of serotonin on the formation of inositol phosphates and protein phosphorylation were examined in cultured smooth muscle cells. Serotonin stimulated the formation of [³H]inositol monophosphate, [³H]inositol bisphosphate and [³H]inositol trisphosphate. This effect was prevented by 5-HT₂ specific antagonist, 6-methyl-1-(1-methylethyl)ergoline-8-carboxylic acid, 2-hydroxy-1-methylpropyl ester [Z]-2-butenedioate (LY53857). Serotonin stimulated the phosphorylation of many polypeptides, among which a 20 kDa polypeptide was the most prominent. The phosphorylation was also inhibited by LY53857. LY53857 alone produced no effects on protein phosphorylation. The 20 kDa polypeptides were also phosphorylated by the addition of 12-O-tetradecanoylphorbol-13-acetate. These results suggest that serotonin stimulates protein phosphorylation through 5-HT₂ receptors and possibly activates protein kinase C in intact vascular smooth muscle cells.Part of the data contained in this paper was presented at the 74th local meeting of the Japanese Society of Pharmacology at Kanagawa.     

The halophilic properties of pyruvate kinase fromVibrio costicola,a moderate halophile

Summary Pyruvate kinase fromVibrio costicola, a moderate halophile, appears to be adapted to functioning in the presence of salt. Its stability depends on the ionic strength of the medium. The amino acid composition resembles that of other halophilic enzymes. It is proposed that the halophilic pyruvate kinase utilizes preferentially the Mn⁺⁺ cofactor which forms more stable complexes in the presence of physiological concentration of salt.     

Zur Biosynthese des Verbenalins und Aucubins

Summary Verbena officinalis specifically incorporates 2-¹⁴C labelled mevalonic acid in the formation of the aglycone moiety of verbenalin (2). In aucubin (6), isolated fromVerbascum thapsus, the incorporation rate is very low and the main part of the activity is located in the sugar moiety.     

Spermine protects protein kinase C from phospholipid-induced inactivation

Phosphatidylserine (PS), an activator of protein kinase C (PKC) in the assay of protein phosphorylation, inhibited this enzyme in a time-dependent manner following preincubation in the absence of Ca²⁺. The phospholipid-induced inactivation of kinase activity was dependent on the PS content and on the charge density of liposomes. This inactivation of PKC could be reduced, but not completely eliminated, by addition of Ca²⁺. In the present work the effect of a naturally occurring polyamine (spermine) on the PS-induced inactivation of PKC was investigated. The presence of spermine during preincubation without Ca²⁺ was effective in suppressing the PS-induced inactivation of PKC over the period (20 min) required for PS to inhibit the enzyme by 95%. PKC exists in two membrane-bound states: a reversible one which can be dissociated by Ca²⁺ chelators (membrane-associated form) and an irreversible one which is chelator-stable (membrane-inserted form). Gel filtration experiments on the PKC-PS complex formed in the presence of Ca²⁺ indicated that less insertion of enzyme into liposomes occurred in the presence of spermine and that the kinase activity of the reversibly membrane-associated PKC was protected from PS inactivation.     

Differential water permeability and regulation of three aquaporin 4 isoforms

Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K⁺ concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.     

Ecdysteroid conjugates in the ovaries of the silkworm,Bombyx mori: 3-phosphates of 2,22-dideoxy-20-hydroxyecdysone and of bombycosterol

Summary Two novel ecdysteroid conjugates, 2,22-dideoxy-20-hydroxyecdysone 3-phosphate (1) and bombycosterol 3-phosphate (2), as well as four known ecdysteroid 22-phosphate esters, have been isolated and characterized from the ovaries of the silkworm,Bombyx mori.     

Neuer Beitrag zur Determination der Imaginalscheiben beiDrosophila

Summary The temperature effective period for the formation of double antennæ and palpi in the gene-combinationsc ec ct; Dfd ^r-L ends with the second half of the third larval stage. The t.e.p. of the mutantsaristopedia andproboscipedia ends with puparium formation. Thus we have another proof that the imaginal discs ofDrosophila are not definitely determined in the last larval stage.     

Unique evolution of Bivalvia arginine kinases

The clams Pseudocardium, Solen, Corbicula and Ensis possess a unique form of arginine kinase (AK) with a molecular mass of 80 kDa and an unusual two-domain structure, a result of gene duplication and subsequent fusion. These AKs also lack two functionally important amino acid residues, Asp⁶² and Arg¹⁹³, which are strictly conserved in other 40-kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. However, these AKs show higher enzyme activity. The cDNA-derived amino acid sequences of 40-kDa AKs from the blood clam Scapharca broughtonii and the oyster Crassostrea gigas were determined. While Asp⁶² and Arg¹⁹³ are conserved in Scapharca AK, these two key residues are replaced by Asn and Lys, respectively, in Crassostrea AK. The native enzyme from Crassostrea and both of the recombinant enzymes show an enzyme activity similar to that of two-domain clam AKs and at least twofold higher than that of other molluskan AKs. Although the replacement of Asp⁶² or Arg¹⁹³ by Gly in normal AK causes a considerable decrease in V_max (6–15% of wild-type enzyme) and a two- to threefold increase in K_m for arginine, the same replacement in Scapharca AK had no pronounced effect on enzyme activity. Together with the observation that bivalve AKs are phylogenetically distinct from other molluskan AKs, these results suggest that bivalve AKs have undergone a unique molecular evolution; the characteristic stabilizing function of residues 62 and 193 has been lost and, consequently, the enzyme shows higher activity than normal.Received 14 October 2003; accepted 1 November 2003     

Analysis of <Emphasis Type="Italic">Dictyostelium</Emphasis> TACC reveals differential interactions with CP224 and unusual dynamics of <Emphasis Type="Italic">Dictyostelium</Emphasis> microtubules

We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dynamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-α-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers.     

Influence of phosphorus supply and light intensity on mycorrhizal response inPisum-Rhizobium-Glomus symbiosis

The influence of mycorrhizal colonization withGlomus mosseae on parameters of N₂ fixation and plant growth was studied in pot experiments with pea plants (Pisum sativum L.) infected withRhizobium leguminosarum and supplied with varied levels of phosphorus (P) and nitrogen (N). Reduced light intensities were used to evaluate the dependence of the microsymbionts on assimilate supply. In plants grown with low P supply, mycorrhization increased the concentration of P in shoots, and thus N₂ fixation. Reduced light intensity significantly depressed mycorrhizal colonization and nodule growth in low-P plants. When P supply did not limit plant growth and N₂ fixation, however, the percentage of mycorrhizal colonization was reduced due to the higher P status, and the microsymbionts were not impaired by low light intensities. To maximize carbohydrate supply, another experiment was carried out at high light intensity of 900 mol m^–2s^–1 and with non-limiting P supply. Nitrogen fertilization, given as starter N, enhanced plant growth, but delayed nodule formation. Towards flowering, nodulation rapidly increased, but less so inGlomus inoculated plants. After 28 days mycorrhizal plants were lower in shoot dry weight, nodule dry weight and nitrogenase activity. The results suggest that under many, but not all, environmental conditions the host plant is able to restrict mycorrhizal colonization and, thus, to prevent impairment ofRhizobium symbiosis.deceased in May 1994     

La voie du pentose phosphate dans le métabolisme du glucose et son interaction avec la glutathion-réductase dans les noyaux de foie de rat

Summary Rat liver nuclei, isolated in sucrose medium, convert carbon-1 of glucose-6-phosphate to CO₂ via the 6-phosphogluconic acid oxidative pathway. Conversion of glucose-1-¹⁴C to¹⁴CO₂ by the cytoplasmic fraction is greatly inhibited by nuclei. In the presence of low concentrations of NADP, the CO₂ formed by nuclei is markedly increased by the addition of GSSG.     

Über die Orientierung bei der Kondensation von Benzil mit monosubstituierten Guanidinen

Summary Condensation of benzil with two monosubstituted guanidines in the presence of 50 mol% of potassium hydroxide leads to the formation of 2-(subst. imino)-4, 4-diphenyl-imidazolidin-5-ones (Ia, b); by reduction of the potassium hydroxide used the formation of these products is more or less driven back in favour of the formation of 3-substituted 5, 5-diphenyl-glycocyamidines (Ic, d). A possible explanation for these orientation phenomena is given.

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5. Mitteilung über Hydantoine, Thiohydantoine und Glykocyamidine².

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4. Mitteilung sieheK. Lempert undJ. Breuer Chem. Ber.92, 1710 (1959).     

Forecasting for the generation of trading signals in financial markets

In this paper we show that optimal trading results can be achieved if we can forecast a key summary statistic of future prices. Consider the following optimization problem. Let the return r_i (over time i=1, 2, ..., n) for the ith day be given and the investor has to make investment decision d_i on the ith day with d_i=1 representing a ‘long' position and d_i=0 a ‘neutral' position. The investment return is given by r=Σⁿ_i=1r_id_i−cΣⁿ⁺¹_i=1∣d_i−d_i−1∣, where c is the transaction cost. The mathematical programming problem of choosing d₁, ..., d_n to maximize r under a given transaction cost c is shown to have an analytic solution, which is a function of a key summary statistic called the largest change before reversal. The largest change before reversal is recommended to be used as an output in a neural network for the generation of trading signals. When neural network forecasting is applied to a dataset of Hang Seng Index Futures Contract traded in Hong Kong, it is shown that forecasting the largest change before reversal outperforms the k‐step‐ahead forecast in achieving higher trading profits. Copyright © 2000 John Wiley & Sons, Ltd.