Synthesis and Secretion of Hydroxyproline-containing Macromolecules in Carrots: II. In vivo Conversion of Peptidyl Proline to Peptidyl Hydroxyproline

The chelator α,α′-dipyridyl prevents the formation of protein-bound hydroxyproline without affecting protein synthesis as measured by the incorporation of ¹⁴C-proline. The inhibitory effect can be overcome by exogenous ferrous ions. Neither α,α′-dipyridyl nor Fe²⁺ interferes in any other known way with the synthesis and secretion of the hydroxyproline-rich proteins normally found in the cell wall. This reversal by Fe²⁺ of the inhibition of proline hydroxylation by α,α′-dipyridyl is used for a study in vivo of the hydroxylation reaction. This reaction has a temperature coefficient of 2.2, suggesting that it is an enzymatic process. Reversal by Fe²⁺ of the α,α′-dipyridyl inhibition can occur after protein synthesis has been arrested with cycloheximide, indicating that peptidyl proline is the substrate of the hydroxylation reaction. Hydroxylation occurs in the cytoplasm, and not in the cell wall, and only for a limited time after the incorporation of proline into the polypeptide chain. This suggests a spatial separation of the enzyme and the substrate.     

Phytochrome Control of Cell Wall-bound Hydroxyproline Content in Etiolated Pea Epicotyls

The red light inhibition of growth of the intact pea (Pisum sativum L. cv. Alaska) third internode was correlated with an increase in the content of cell wall-bound hydroxyproline. These changes were detected 3 hours after irradiation, and possibly at 1 hour. Far red light reversed the effects of red light. The iron chelator α,α′-dipyridyl reversed the red light effects on both growth and hydroxyproline content. Using segments incubated in vitro, no phytochrome-mediated change in hydroxyproline content could be observed, perhaps because of an overwhelming wounding response. If plants were irradiated in situ and grown for 8 hours before excision and incubation of segments, some enhancement of hydroxylation by red light was detectable both colorimetrically and radioisotopically. The red light inhibition of segment growth was reversed by α,α′-dipyridyl. These results are examined in reference to the role of extensin in normal and induced growth cessation.     

Apparent Inhibition of beta-Fructosidase Secretion by Tunicamycin May Be Explained by Breakdown of the Unglycosylated Protein during Secretion

Suspension-cultured carrot (Daucus carota) cells synthesize and secrete β-fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of β-fructosidase as measured by the accumulation of the radioactive protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated β-fructosidase is synthesized and is associated with an endoplasmic-reticulum-rich microsomal fraction. Pulse-chase experiments show that the unglycosylated β-fructosidase does not remain in the cells and appears to be secreted in the same way as glycosylated β-fructosidase; however, no radioactive, unglycosylated β-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete β-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated β-fructosidase. In the presence of tunicamycin, there is no accumulation of β-fructosidase activity or unglycosylated β-fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of β-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall.     

The Metabolism of Oat Leaves during Senescence: IV. The Effects of alphaalpha'-Dipyridyl and other Metal Chelators on Senescence

The senescence of the first leaves of light-grown Avena seedlings when detached and placed in the dark is inhibited by α, α′-dipyridyl and α, α′, α″-tripyridyl at concentrations between 10⁻⁵ and 10⁻⁴ M. Five other chelating agents exert similar inhibiting effects at concentrations 3 to 30 times higher. The senescence of etiolated leaves, as shown by loss of carotenoid and protein, is similarly inhibited. Ethylene-diaminetetraacetate has a similar effect in the dark, though only at 10 mM and above, but in the light it causes bleaching of chlorophyll. It is deduced that an iron-containing system plays an essential part in the initiation of the senescence process.     

Characterization of Carrot Cell Wall Protein : II. IMMUNOLOGICAL STUDY OF CELL WALL PROTEIN

Antiserum was raised against a synthetic nona-peptide which was predicted to have considerable homology with the unhydroxylated, unglycosylated precursor of cell wall proteins from several plants. The antiserum is able to recognize the major cell wall protein of incubated carrot (Daucus carota) root discs which is produced when the discs are treated with a proline hydroxylase inhibitor and then labeled with radioactive proline. This technique has potential applications in studying cell wall biosynthesis and its regulatory control mechanisms.     

A 3-hydroxy β-end group in xanthophylls is preferentially oxidized to a 3-oxo ε-end group in mammals

We previously found that mice fed lutein accumulated its oxidative metabolites (3′-hydroxy-ε,ε-caroten-3-one and ε,ε-carotene-3,3′-dione) as major carotenoids, suggesting that mammals can convert xanthophylls to keto-carotenoids by the oxidation of hydroxyl groups. Here we elucidated the metabolic activities of mouse liver for several xanthophylls. When lutein was incubated with liver postmitochondrial fraction in the presence of NAD⁺, (3′R,6′R)-3′-hydroxy-β,ε-caroten-3-one and (6RS,3′R,6′R)-3′-hydroxy-ε,ε-caroten-3-one were produced as major oxidation products. The former accumulated only at the early stage and was assumed to be an intermediate, followed by isomerization to the latter. The configuration at the C3′ and C6′ of the ε-end group in lutein was retained in the two oxidation products. These results indicate that the 3-hydroxy β-end group in lutein was preferentially oxidized to a 3-oxo ε-end group via a 3-oxo β-end group. Other xanthophylls such as β-cryptoxanthin and zeaxanthin, which have a 3-hydroxy β-end group, were also oxidized in the same manner as lutein. These keto-carotenoids, derived from dietary xanthophylls, were confirmed to be present in plasma of normal human subjects, and β,ε-caroten-3′-one was significantly increased by the ingestion of β-cryptoxanthin. Thus, humans as well as mice have oxidative activity to convert the 3-hydroxy β-end group of xanthophylls to a 3-oxo ε-end group.     

Gemfibrozil,a Lipid-lowering Drug,Increases Myelin Genes in Human Oligodendrocytes via Peroxisome Proliferator-activated Receptor-β

An increase in CNS remyelination and a decrease in CNS inflammation are important steps to halt the progression of multiple sclerosis. Earlier studies have shown that gemfibrozil, a lipid-lowering drug, has anti-inflammatory properties. The current study identified another novel property of gemfibrozil in stimulating the expression of myelin-specific genes (myelin basic protein, myelin oligodendrocyte glycoprotein, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, and proteolipid protein (PLP)) in primary human oligodendrocytes, mixed glial cells, and spinal cord organotypic cultures. Although gemfibrozil is a known activator of peroxisome proliferator-activated receptor-α (PPAR-α), we were unable to detect PPAR-α in either gemfibrozil-treated or untreated human oligodendrocytes, and gemfibrozil increased the expression of myelin genes in oligodendrocytes isolated from both wild type and PPAR-α(−/−) mice. On the other hand, gemfibrozil markedly increased the expression of PPAR-β but not PPAR-γ. Consistently, antisense knockdown of PPAR-β, but not PPAR-γ, abrogated the stimulatory effect of gemfibrozil on myelin genes in human oligodendrocytes. Gemfibrozil also did not up-regulate myelin genes in oligodendroglia isolated from PPAR-β(−/−) mice. Chromatin immunoprecipitation analysis showed that gemfibrozil induced the recruitment of PPAR-β to the promoter of PLP and myelin oligodendrocyte glycoprotein genes in human oligodendrocytes. Furthermore, gemfibrozil treatment also led to the recruitment of PPAR-β to the PLP promoter in vivo in the spinal cord of experimental autoimmune encephalomyelitis mice and suppression of experimental autoimmune encephalomyelitis symptoms in PLP-T cell receptor transgenic mice. These results suggest that gemfibrozil stimulates the expression of myelin genes via PPAR-β and that gemfibrozil, a prescribed drug for humans, may find further therapeutic use in demyelinating diseases.     

Characterization of a xylose-specific antiserum that reacts with the complex asparagine-linked glycans of extracellular and vacuolar glycoproteins

Antibodies were raised against carrot (Daucus carota) cell wall β-fructosidase that was either in a native configuration (this serum is called anti-βF₁) or chemically deglycosylated (anti-βF₂). The two antisera had completely different specificities when tested by immunoblotting. The anti-βF₁ serum reacted with β-fructosidase and many other carrot cell wall proteins as well as with many proteins in extracts of bean (Phaseolus vulgaris) cotyledons and tobacco (Nicotiana tabacum) seeds. It did not react with chemically deglycosylated β-fructosidase. The anti-βF₁ serum also reacted with the bean vacuolar protein, phytohemagglutinin, but not with deglycosylated phytohemagglutinin. The anti-βF₂ serum reacted with both normal and deglycosylated β-fructosidase but not with other proteins. These results indicate that the βF₂ antibodies recognize the β-fructosidase polypeptide, while the βF₁ antibodies recognize glycan sidechains common to many glycoproteins. We used immunoadsorption on glycoprotein-Sepharose columns and hapten inhibition of immunoblot reactions to characterize the nature of the antigenic site. Antibody binding activity was found to be associated with Man₃(Xyl)(GIcNAc)₂Fuc, Man₃(Xyl)(GIcNAc)₂, and Man(Xyl) (GIcNAc)₂ glycans, but not with Man₃(GIcNAc)₂. Treatment of phytohemagglutinin, a glycoprotein with a Man₃(Xyl)(GIcNAc)₂Fuc glycan, with Charonia lampas β-xylosidase (after treatment with jack-bean α-mannosidase) greatly diminished the binding between the antibodies and phytohemagglutinin. We conclude, therefore, that the antibodies bind primarily to the xyloseβ, 1→ 2mannose structure commonly found in the complex glycans of plant glycoproteins.     

Identification and characterization of a novel testis-specific gene CKT2, which encodes a substrate for protein kinase CK2

Protein kinase CK2 is a serine/threonine kinase known to phosphorylate numerous substrates. CK2 is implicated in several physiologic and pathologic processes, particularly in cancer biology. CK2 is comprised of several subunits, including CK2α, CK2α′ and CK2β. Inactivation of CK2α′ leads to chromatin degeneration of germ cells, resulting in male sterility. To identify additional targets of CK2α′ in testes and to determine the role of CK2α′ in germ cell nuclear integrity, GST pull-down and protein–protein interaction assays were conducted. A novel testis-specific gene, CKT2 (CK2 Target protein 2), was found whose product interacts with and is phosphorylated by CK2 in vitro and in vivo. CKT2 is a 30.2 kDa protein with one coiled-coil domain and six putative phosphorylation sites. High expression of CKT2 correlated with chromatin condensation of spermatids in murine testes. Findings reported herein demonstrate that CKT2 is a target protein of native CK2α′ in testes and suggest that CKT2 plays a role in chromatin regulation of male germ cells.     

Effects of sulfur nutrition on expression of the soybean seed storage protein genes in transgenic petunia

The 7S seed storage protein (β-conglycinin) of soybean (Glycine max [L]. Merr.) has three major subunits; α, α′, and β. Accumulation of the β-subunit, but not the α- and α′-subunits, has been shown to be repressed by exogenously applied methionine to the immature cotyledon culture system (LP Holowach, JF Thompson, JT Madison [1984] Plant Physiol 74: 576-583) and to be enhanced under sulfate deficiency in soybean plants (KR Gayler, GE Sykes [1985] Plant Physiol 78: 582-585). Transgenic petunia (Petunia hybrida) harboring either the α′- or β-subunit gene were constructed to test whether the patterns of differential expression were retained in petunia. Petunia regulates these genes in a similar way as soybean in response to sulfur nutritional stimuli, i.e. (a) expression of the β-subunit gene is repressed by exogenous methionine in in vitro cultured seeds, whereas the α′-subunit gene expression is not affected; and (b) accumulation of the β-subunit is enhanced by sulfur deficiency. The pattern of accumulation of major seed storage protein of petunia was not affected by these treatments. These results indicate that this mechanism of gene regulation in response to sulfur nutrition is conserved in petunia even though it is not used to regulate its own major seed storage proteins.     

Turnover of 1-aminocyclopropane-1-carboxylic Acid synthase protein in wounded tomato fruit tissue

Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [³⁵S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins.     

Additive Expression of Consolidated Memory through Drosophila Mushroom Body Subsets

Associative olfactory memory in Drosophila has two components called labile anesthesia-sensitive memory and consolidated anesthesia-resistant memory (ARM). Mushroom body (MB) is a brain region critical for the olfactory memory and comprised of 2000 neurons that can be classified into αβ, α′β′, and γ neurons. Previously we demonstrated that two parallel pathways mediated ARM consolidation: the serotonergic dorsal paired medial (DPM)–αβ neurons and the octopaminergic anterior paired lateral (APL)–α′β′ neurons. This finding prompted us to ask how this composite ARM is retrieved. Here, we showed that blocking the output of αβ neurons and that of α′β′ neurons each impaired ARM retrieval, and blocking both simultaneously had an additive effect. Knockdown of radish and octβ2R in αβ and α′β′ neurons, respectively, impaired ARM. A combinatorial assay of radish mutant background rsh¹ and neurotransmission blockade confirmed that ARM retrieved from α′β′ neuron output is independent of radish. We identified MBON-β2β′2a and MBON-β′2mp as the MB output neurons downstream of αβ and α′β′ neurons, respectively, whose glutamatergic transmissions also additively contribute to ARM retrieval. Finally, we showed that α′β′ neurons could be functionally subdivided into α′β′m neurons required for ARM retrieval, and α′β′ap neurons required for ARM consolidation. Our work demonstrated that two parallel neural pathways mediating ARM consolidation in Drosophila MB additively contribute to ARM expression during retrieval.     

Expression of Intracellular Interferon-Alpha Confers Antiviral Properties in Transfected Bovine Fetal Fibroblasts and Does Not Affect the Full Development of SCNT Embryos

Foot-and-mouth disease, one of the most significant diseases of dairy herds, has substantial effects on farm economics, and currently, disease control measures are limited. In this study, we constructed a vector with a human interferon-α (hIFN-α) (without secretory signal sequence) gene cassette containing the immediate early promoter of human cytomegalovirus. Stably transfected bovine fetal fibroblasts were obtained by G418 selection, and hIFN-α transgenic embryos were produced by somatic cell nuclear transfer (SCNT). Forty-six transgenic embryos were transplanted into surrogate cows, and five cows (10.9%) became pregnant. Two male cloned calves were born. Expression of hIFN-α was detected in transfected bovine fetal fibroblasts, transgenic SCNT embryos, and different tissues from a transgenic SCNT calf at two days old. In transfected bovine fetal fibroblasts, expression of intracellular IFN-α induced resistance to vesicular stomatitis virus infection, increased apoptosis, and induced the expression of double-stranded RNA-activated protein kinase gene (PKR) and the 2′-5′-oligoadenylate synthetase gene (2′-5′ OAS), which are IFN-inducible genes with antiviral activity. Analysis by qRT-PCR showed that the mRNA expression levels of PKR, 2′-5′ OAS, and P53 were significantly increased in wild-type bovine fetal fibroblasts stimulated with extracellular recombinant human IFN-α-2b, showing that intracellular IFN-α induces biological functions similar to extracellular IFN-α. In conclusion, expression of intracellular hIFN-α conferred antiviral properties in transfected bovine fetal fibroblasts and did not significantly affect the full development of SCNT embryos. Thus, IFN-α transgenic technology may provide a revolutionary way to achieve elite breeding of livestock.     

Transformation of 2,2′-Bimorphine to the Novel Compounds 10-α-S-Monohydroxy-2,2′-Bimorphine and 10,10′-α,α′-S,S′-Dihydroxy-2,2′-Bimorphine by Cylindrocarpon didymum

Whole-cell suspensions of Cylindrocarpon didymum were observed to transform 2,2′-bimorphine to the compounds 10-α-S-monohydroxy-2,2′-bimorphine and 10,10′-α,α′-S,S′-dihydroxy-2,2′-bimorphine. Mass spectrometry and ¹H nuclear magnetic resonance spectroscopy confirmed the identities of these new morphine alkaloids.     

Cyanide-insensitive Respiration in Plant Mitochondria

Pathways of electron transport have been studied in mitochondria isolated from hypocotyls of etiolated mung bean seedlings and skunk cabbage spadices that show cyanide-resistant respiratory activity. The residual flux through cytochrome c oxidase is shown to be small in comparison with the flux through an unidentified alternative oxidase that is known to have a high affinity for oxygen. This alternative oxidase is not a cytochrome. Skunk cabbage and mung bean mitochondria contain cytochromes a and a₃ that have absorption peaks differing slightly from those of animal preparations. A slow oxidation-reduction of cytochrome a₃-CN has been demonstrated. Cytochromes b undergo oxidation and reduction in the presence of cyanide but play no essential role in the cyanide-resistant pathway. Antimycin inhibits to an extent similar to that of cyanide; the respiratory chain bifurcates on the substrate side of the antimycin-sensitive site. Evidence is presented for the selective inhibition by thiocyanate, α, α′-dipyridyl, and 8-hydroxyquinoline of the alternative oxidase pathway, which may therefore contain a non-heme iron protein.     

Microbial 7α-Hydroxylation of 3-Ketobisnorcholenol

The transformation of 22-hydroxy-23,24-bisnorchol-4-en-3-one to 7α-22-dihydroxy-23,24-bisnorchol-4-en-3-one by Botryodiploida theobromae, Lasiodiplodia theobromae, and various Botryosphaeria strains is described. Factors affecting the reaction were incubation temperature, sonication of the substrate, and addition of 2,2′-dipyridyl, extra carbohydrate, and Amberlite XAD-7. The enzyme responsible for the reaction appeared to be very specific and was not characteristic of all members of the genera listed above.     

Analysis of the β-1,3-Glucanolytic System of the Biocontrol Agent Trichoderma harzianum

The biocontrol agent Trichoderma harzianum IMI206040 secretes β-1,3-glucanases in the presence of different glucose polymers and fungal cell walls. The level of β-1,3-glucanase activity secreted was found to be proportional to the amount of glucan present in the inducer. The fungus produces at least seven extracellular β-1,3-glucanases upon induction with laminarin, a soluble β-1,3-glucan. The molecular weights of five of these enzymes fall in the range from 60,000 to 80,000, and their pIs are 5.0 to 6.8. In addition, a 35-kDa protein with a pI of 5.5 and a 39-kDa protein are also secreted. Glucose appears to inhibit the formation of all of the inducible β-1,3-glucanases detected. A 77-kDa glucanase was partially purified from the laminarin culture filtrate. This enzyme is glycosylated and belongs to the exo-β-1,3-glucanase group. The properties of this complex group of enzymes suggest that the enzymes might play different roles in host cell wall lysis during mycoparasitism.     

Selective Solubilization of Membrane Proteins Differentially Labeled by p-Chloromercuribenzenesulfonic Acid in the Presence of Sucrose

Broadbean (Vicia faba L.) leaf discs have been incubated with the slowly permeant thiol reagent [²⁰³Hg]-para-chloromercuribenzenesulfonic acid (PCMBS) in the presence or in the absence of sucrose, and the release of PCMBS-labeled proteins has been monitored in media containing various concentrations of urea, ethyleneglycol-bis-(β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), sodium cholate, sodium dodecyl sulfate, Triton X-100, octylglucoside or (3-[3-cholamidopropyl)-dimethylammonio] 1-propane-sulfonate) (CHAPS). The proteins differentially labeled by PCMBS in the presence of sucrose which, on the basis of previous results, are assumed to include the sucrose carrier, were preferentially solubilized by 1% CHAPS, 1% octylglucoside, or 1% Triton X-100. Other PCMBS-labeled proteins (`background' proteins) could be partially removed by EGTA, urea, or 0.1% cholate. Sequential treatment by 10 mm EGTA and 1% CHAPS was found to give a fraction highly enriched in the differentially labeled proteins. Analysis of the specific activity of microsomal pellets suggests that the results obtained with leaf discs give a good account of what is occurring at the plasma membrane level. These data, which suggest that the proteins differentially labeled by PCMBS in the presence of sucrose are intrinsic membrane proteins, can be used to solubilize these proteins from microsomal fractions.