Degradation products of myelin proteins in a light CNS subcellular fraction

The fraction floating on 0.32 M sucrose when normal mammalian spinal cord homogenate is submitted to discontinuous density gradient centrifugation is highly enriched in Marchi-positive material. In situ this material is located along paranodal myelin sheath segments. We here show by immunoblotting that degradation products of the myelin-associated glycoprotein (MAG) and of the enzyme 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) is present in the Marchi-positive floating fraction but is not found in the myelin fraction. Since previous biochemical analyses of the floating fraction show a gross composition closely resembling myelin and since metabolic studies show the specific activity of incorporated amino acids to proceed with time from beavier to lighter myelin subfractions the results strongly suggest that normally occurring Marchi-positive bodies represents an intermediate stage in myelin catabolism.     

Serotoninergic innervation of the alimentary canal of the Colorado potato beetle,Leptinotarsa decemlineata: structural and functional aspects

Phenol (aryl) sulfotransferases (PSTs) provide a conjugative pathway that detoxifies hydroxylated aromatic xenobiotics by esterification with sulfate. Both human and bovine airways have been reported to use this pathway, and in this investigation the bovine system is examined. PST activity in tracheal through fourth generation bronchial mucosal cytosols was 0.1–0.35 nmol/mg protein/min. Activity was generally greater in more distal bronchi and in parenchymal extracts, which contained 0.6–3 nmol/mg/min PST activity. Comparison of the PST activities of bronchial and parenchymal cytosols indicated similar pH activity profiles, although steady state kinetic measurements revealed different K_m values for the acceptor substrate 2-naphthol (13.7 M for bronchial, 31.3 M for parenchymal). Anion exchange chromatography indicated two PST isoforms being expressed in different ratios. Immunoblot analysis with mouse antibovine PST revealed a closely spaced doublet at 32 kDa in both bronchial mucosal and parenchymal cytosolic extracts; however, this doublet was unequally stained in parenchymal extracts. Immunohistochemical analyses revealed faint positive staining of the tracheobronchial epithelium. Greatest immunostaining was observed in the nonciliated secretory epithelial cells of the bronchioles, whereas surrounding smooth muscle, endothelial cells, and alveoli were immunonegative. These results are consistent with the known locations of other detoxification enzymes within the airways.     

AMP deaminase in Dictycostelium discoideum: Increase in activity following nutrient deprivation induced by starvation or hadacidin

Summary AMP deaminase, the activity that catalyzes the deamination of AMP to form IMP and NH₃ has been measured in Dictyostelium discoideum. A new procedure to assay the activity of this enzyme was developed using formycin 5-monophosphate, a fluorescent analog of AMP as the substrate, and ionpaired reverse phase HPLC to separate the reactants and products. Quantitation of the formycin containing compounds was accomplished at 290 nm. At this wavelength adenosine containing compounds were not detected and activity could be monitored in the presence of its activator ATP. The AMP deaminase activity in vegetative cells was 7.4 nmols/min/mg proteins while the activity in cells measured at 2 and 6 hrs after starvation-induced growth-arrest was 376 nmols/min/mg protein... a 51-fold increase. When vegetative cells were treated with hadacidin, a drug that restricts de novo AMP synthesis and pinocytosis, the activity of the AMP deaminase was 511 nmols/min/mg protein... a 70-fold increase compared to that in untreated vegetative cells. Smaller increases were noted following the inhibition of growth with the drugs cerulenin and vinblastine, as well as after the inhibition of de novo GMP synthesis with the drug mycophenolic acid or the partial inhibition of de novo AMP synthesis with analogs of hadacidin, N-hydroxyglycine and N-formylglycine. In addition, when the activity of two other enzymes involved in purine metabolism, namely adenosine kinase and hypoxanthine-guanine phosphoribosyl transferase, was measured in vegetative cells, and the activity of both compared to that measured in starvation and hadacidin induced growth-arrested cells, showed no significant changes. These data suggest that the changes in the activity of the AMP deaminase are in response to nutrient deprivation and further, that as a consequence of the increase in AMP deaminase activity, ammonia will be produced and an increase in pH should follow. The production of ammonia and its effect on development implicates the AMP deaminase in the early differentiation of this organism.     

Alternative splicing of pre-mRNA encoding the <Emphasis Type="Italic">Musca domestica</Emphasis> latrophilin-like protein: Primary structures of four spliced forms of mRNA and their protein products

An internal DNA fragment (2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5- and 3-ends (5- and 3-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (6000 nt) differ in the structures of both the regions encoding signal peptides and 5-terminal untranslated regions. They encode large proteins (1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane strands followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severely truncated at their C-termini (900 aa). They are composed of the domain with only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 175–185.Original Russian Text Copyright © 2005 by Andreev, Danilevich, Grishin.     

DeoxyNAD and deoxyADP-ribosylation of proteins

Recently, two deoxyribose analogs of NAD⁺ (2-deoxy and 3-deoxyNAD⁺) have been synthesized and purified in this laboratory. Whereas 2-deoxyNAD⁺ was an efficient substrate for arg-specific mon(ADP-ribosyl) transferases, it was not a substrate for poly(ADP-ribose) polymerase (PARP). Instead, it was a non-competitive inhibitor of NAD⁺ in the ADP-ribose polymerization reaction catalyzed by PARP. Thus, 2-deoxyNAD⁺ has been utilized to distinguish between mono(ADP-ribose) and poly(ADP-ribose) acceptor proteins. 2-deoxyNAD⁺ has also been used to characterize the arg-specific mono(2-deoxyADP-ribosyl)ation reaction of PARP with cholera toxin or avian mono(ADP-ribosyl)transferase. By contrast, 3-deoxyNAD⁺ can effectively be utilized as a substrate by PARP. However, while the estimated Km and Kcat of polymerization with 3-deoxyNAD⁺ can were 20 M and 0.11 moles/sec, the Km and Kcat with NAD⁺ as a substrate were 59 M and 1.29 moles/sec, respectively. Determination of the average size of 3-deoxyADP-ribose polymers indicated that chains no larger than four residues are synthesized with this substrate. Thus, the utilization of 3-deoxyNAD⁺ has facilitated the electrophoretic identification of poly(ADP-ribose) acceptor proteins in mammalian chromatin.     

The mitochondrial inner membrane anion channel is inhibited by DIDS

The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg²⁺ and H⁺. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2-disulfonates. Inhibition of malonate transport approaches 100% with IC₅₀ values of 26, 44, and 88 M for DIDS, H₂-DIDS, and SITS respectively and Hill coefficients 1. In contrast, inhibition of Cl^– transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC₅₀ which is severalfold higher than that for malonate. The IC₅₀ for malonate transport is decreased about 50% by pretreatment of the mitochondria withN-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC₅₀ by about 50%, but under conditions where only the matrix pH is made alkaline the IC₅₀ is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other Cl^– channels than previously thought, but also suggest that other Cl^– channels may be sensitive to some of the many regulators of IMAC which have been identified.     

Cofactor interactions and the regulation of glutamate decarboxylase activity

More than 50% of glutamate decarboxylase (GAD) in brain is present as apoenzyme. Recent work has opened the possibility that apoGAD can be studied in brain by labeling with radioactive cofactor. Such studies would be aided by a compound that inhibits specific binding. One possibility is 4-deoxy-pyridoxine 5-phosphate, a close structural analog of the cofactor pyridoxal 5-phosphate. The effects of deoxypyridoxine-P on the cyclic series of reactions that interconverts apo- and holoGAD was investigated and found to be consistent with simple competitive inhibition of the activation of apoGAD by pyridoxal-P. As expected from the cycle GAD was inactivated when incubated with glutamate and deoxypyridoxine-P even though cofactor was present, but no inactivation was observed with deoxypyridoxine-P in the absence of glutamate. Deoxypyridoxine-P also stabilized apoGAD against heat denaturation. These effects were quantitatively accounted for by a kinetic model of the apo-holoGAD cycle. Deoxypyridoxine-P inhibited the labeling by [³²P]pyridoxal-P of GAD isolated from rat brain. Hippocampal extracts were labeled with [³²P]pyridoxal-P and analyzed by SDS-polyacrylamide gel electrophoresis. Remarkably few bands were strongly labeled. The major labeled band (at 63 kDa) corresponded to one of the forms of GAD. Other strongly-labeled bands were observed at 65 kDa (corresponding to the higher molecular weight form of GAD) and at 69–72 kDa. Labeling of the 63- and 65-kDa bands was inhibited by deoxypyridoxine-P, but the 69–72 kDa bands were unaffected, suggesting that the latter were non-specifically labeled. The results suggest that the 63-kDa form of GAD makes up the majority of apoGAD in hippocampus.Special issue dedicated to Dr. Eugene Roberts.     

Monitoring the mitochondrial transmembrane potential with the JC-1 fluorochrome in programmed cell death during mesophyll leaf senescence

Summary. Analysis of the mitochondrial transmembrane potential (_m) with the help of the JC-1 fluorochrome (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide) during mesophyll leaf senescence was performed in order to determine whether a reduction of _m takes place during mesophyll senescence and whether plant mitochondria, like mammalian ones, might be involved in the induction of programmed cell death. Fluorescence analysis of mesophyll protoplasts of Pisum sativum in a confocal microscope, fluorescent spectra analysis and time dependence of fluorescence intensity of monomers and of J-aggregates revealed that JC-1 is incorporated and accumulated specifically in plant mitochondria. Analysis of _m during mesophyll protoplast senescence revealed that two subpopulations of mitochondria which differ in _m exist in all analyzed stages of leaf senescence. The first subpopulation contains mitochondria with red fluorescence of J-aggregates due to an unperturbed high _m. The second subpopulation comprises mitochondria with green fluorescence of monomers due to a low _m, proving total depolarization of mitochondrial membranes. Fluorescence analysis demonstrated that even in the latest analyzed stages of leaf senescence, mitochondria with a high _m still exist. Fluorometric measurements revealed that the fluorescence intensity of J-aggregates decreases with the age of plants, which indicates that a reduction of _m during the mesophyll senescence process takes place; however, it does not take place within the whole population of mitochondria of the same protoplast. The reason of this can be due to a dramatic reorganization of mitochondria in mesophyll cells and the appearance of large mitochondria with local heterogeneity of _m in the oldest analyzed stages. All mitochondria in every stage of senescence maintained their membrane organization even when their size, distribution, and spatial organization in protoplasts changed dramatically. We stated that the reduction of _m does not directly induce programmed cell death in mesophyll cells, as opposed to animal apoptosis.Correspondence and reprints: Department of Plant Anatomy and Cytology, Institute of Experimental Biology of Plants, Warsaw University, Miecznikowa 1, 02-096 Warszawa, Poland.     

Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.     

Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers

Anthocyanins are responsible for reds through blues in flowers. Blue and violet flowers generally contain derivatives of delphinidin, whereas red and pink flowers contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes. Flavonoid-3',5'-hydroxylase, a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, generally required for blue or purple flowers. Here we report on the isolation of a cDNA clone of a putative flavonoid-3',5'-hydroxylase gene from Phalaenopsis that was then cloned into a plant expression vector. Transient transformation was achieved by particle bombardment of Phalaenopsis petals. The transgenic petals changed from pink to magenta, indicating that the product of the putative flavonoid-3',5'-hydroxylase gene influences anthocyanin pigment synthesis.     

Xanthoxin levels and metabolism in the wild-type and wilty mutants of tomato

Using ¹³C-labelled internal standards and gas chromatography-mass spectrometry/multiple-ion monitoring the levels of xanthoxin (Xan) and 2-trans-xanthoxin (t-Xan) have been determined in stressed and non-stressed leaves of wildtype tomato (Lycopersicon esculentum Mill cv. Ailsa Craig), and the wilty mutants, notabilis (not), flacca (flc) and sitiens (sit). Levels of Xan were very low in all tissues. Ratios of t-Xan: Xan ranged from 10:1 to <500:1. In the wild-type and flc, t-Xan levels increased following stress. The results from feeding experiments using [¹³C]Xan and t-Xan demonstrated that whilst wild-type and not plants readily converted Xan into abscisic acid (ABA), flc and sit plants converted only a small amount of applied Xan into ABA. In all plants t-Xan was not converted into ABA. These results indicate that the flc and sit mutants are impaired in ABA biosynthesis because they are unable to convert Xan into ABA, whereas the not mutant is blocked at a metabolic step prior to Xan. Another possible ABA precursor, ABA-1,4-trans-diol (ABA-t-diol) was found to occur in wild-type and mutant tissue. All four tissues could convert [²H]ABA-t-diol to ABA. Incubation of stressed leaves in the presence of ¹⁸O₂ provided evidence consistent with Xan and ABA originating via oxidative cleavage of a xanthophyll such as violaxanthin.Abbreviations ABA abscisic acid - ABA-t-diol abscisic acid-1,4-trans-diol - DDC sodium diethyldithiocarbamate - FW fresh weight - GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - MIM multiple-ion monitoring - PA phaseic acid - Xan xanthoxin - flc flacca - not notabilis - sit sitiens     

The use of the luxA gene of the bacterial luciferase operon as a reporter gene

Summary Bacterial luciferase can be assayed rapidly and with high sensitivity both in vivo and in vitro. Here we demonstrate that the N-terminal hydrophobic domain of the catalytic subunit of the luciferase enzyme is indispensable for enzyme activity, although N-terminal translational fusions with full luciferase activity can be obtained. Bacterial luciferase is therefore ideally suited as a reporter enzyme for gene fusion experiments. A list of vectors for the convenient use of the luciferase marker genes to monitor gene expression in vivo are presented.     

The involvement of the 3′:5′-cyclic-AMP phosphodiesterase in transformation and growth of crown-gall tumors in Bryophyllum daigremontianum

In crown-gall tumor tissue obtained from leaves of Bryophyllum daigremontianum an adenosine 3:5-cyclic phosphate (3:5-cyclic-AMP) degrading activity increases up to 2.5 fold until the fifth day after inoculation with Agrobacterium tumefaciens, declining to the value of the control in the solid tumor. Theophylline up to 1 mmol l^–1 given to wounded leaves of Bryophyllum daigremontianum has no effect on the number of tumors. The effect of higher concentrations given over extended periods can be explained otherwise. Therefore it seems likely that the 3:5-cyclic-AMP phosphodiesterase (EC 3.1.4.17) has no effect on transformation and growth of crown-gall tumors in Bryophyllum daigremontianum.     

Characterization and localization of fusicoccin-binding sites in leaf tissues of Vicia faba L. probed with a novel radioligand

Tritiated 9-nor-fusicoccin-8-alcohol provides a highly bioactive radioligand of high specific activity which is easily prepared by oxidation of fusicoccin and subsequent reduction with tritiated sodium borohydride. Using this radioligand, we have identified and characterized a selective binding site for fusicoccin (K_a for [³H]-9-nor-fusicoccin-8-alcohol=0.20·10⁹ M^-1; K_a, apparent for fusicoccin=0.21·10⁹ M^-1) located at the plasmalemma of Vicia faba leaf tissue. The site is thermolabile, readily degraded by trypsin and located at the apoplastic face of the plasmalemma based on results obtained using right-side-out plasmalemma vesicles prepared by aqueous two-phase partitioning and macromolecular fusicoccin-derivatives. The binding-protein is present in guard cells of Vicia faba, as shown by the use of purified guard-cell protoplasts.Abbreviations BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FC fusicoccin - FCol 9-nor-fusicoccin-8-alcohol - Mes 2(N-morpholino)ethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Intracellular calcium activity in split frog skin epithelium: Effect of cAMP

Summary Measurement of intracellular calcium activity (a _Ca ^c ) by ion-selective microelectrodes has previously been technically limited to relatively large cells (20 m). We now report results obtained with this technique in the small epithelial cells (10 m) of split frog skin using microelectrodes having an outer tip diameter of <0.2 m. The basolateral membrane potential was measured with Ca²⁺-selective microelectrodes (E _Ca ^sc ) and with reference micropipettes ( ^sc ) either sequentially or simultaneously in 15 successful experiments. Under baseline conditions,a _Ca ^c was measured to be 215±39nm (mean±se), in close agreement with the mean values estimated from published data obtained withNecturus proximal tubule. Stimulation of Na⁺ transport across six skins with 1mm serosal 8p-chlorophenylthio-3,5 cyclic AMP (CPTcAMP) increaseda _Ca ^c by a factor of 2.6±0.6. The increase ina _Ca ^c preceded the CPTcAMP-induced increase inI _sc. The results of the present study indicate that electrometric determination of intracellular calcium activity is now feasible in a much wider range of cell systems than heretofore possible. CPT cAMP elevates intracellular Ca²⁺ activity; this phenomenon is an early event, preceding the natriferic effect of CPTcAMP.     

Complex Formation of E. coli RNA Polymerase with Bacteriophage T2 DNA: Long-Range Effects in DNA

Conformation behavior of phase T2 DNA in the process of its interaction with it E. coli RNA polymerase was studied using spin labeling technique. T2 DNA was modified by the spin-labeled imidazole at OH-groups of glucosylated cytidine residues. It was shown that the binding of RNA polymerase under the conditions favoring the formation of open promoter complexes induces specific conformational changes in the spin-labeled DNA. The observed conformational changes encompass not only the promoter regions of DNA which are involved in direct contacts with RNA polymerase molecules but extend over remote DNA sites (long-range effect). In relation to this effect, current theoretical models of DNA dynamics are discussed.     

Co-localization of polysomes,cytoskeleton, and membranes with protein bodies from corn endosperm

Summary Frozen corn endosperm was homogenized in a cytoskeleton-stabilizing buffer and stained directly (without pelleting) with rhodamine-phalloidin for actin and either thiazole orange to stain RNA or DiOC₆ to stain membranes prior to examination under the fluorescence microscope. Other samples were treated with a non-ionic detergent alone or in conjunction with a ionic detergent prior to staining and fluorescence microscopy. Very gentle homogenization in unsupplemented buffer yielded a massive aggregate containing protein bodies that fluoresced after treatment with the ER stain DiOC₆. This aggregate was capped by an aggregate of unstained starch grains. More vigorous homogenization yielded more disperse patterns showing almost identical co-localization of ER, actin and RNA (polysomes). Homogenization in buffer plus non-ionic detergent removed most of the membrane yet maintained co-localization of actin and polysomes, while homogenization in double detergent removed the last traces of membrane and actin, and released over 70% of the polysomes. We interpret these results to suggest that protein bodies are surrounded by membranes, cytoskeleton and RNA (polysomes) and that the majority of the polysomes are attached more firmly to the cytoskeleton than to the membrane. This provides evidence from fluorescence microscopy to supplement that from biochemical analyses for the existence of cytomatrix-bound polysomes in plants.Abbreviations CBP cytoskeleton-bound polysomes - CMBP cyto-matrix-bound polysomes - CSB cytoskeleton-stabilizing buffer - DOC sodium deoxycholate - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DTE dithioerythritol - MBP membrane-bound polysomes - FP free polysomes - PMSF phenylmethyl-sulfonyl fluoride - PTE polyoxy-ethylene-10-tridecyl ether - Rh-Ph rhodamine-phalloidin - TO thiazole orange - Tris tris-(hydroxymethyl) aminomethane