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PubMed2002

Chemical polymorphism of the cuticular lipids of the cabbage white Pieris rapae.

The epicuticular composition of different body parts of the Cabbage White, Pieris rapae L., was investigated using GC and GC/MS. The major group of components, hydrocarbons, occurs in two distinct classes, which show different distributions on the cuticle of the insects. Unbranched shorter chain compounds (C21 to C31, linear group) dominate on body, head and wings, while longer chain, polymethyl-branched compounds (C35 to C39, branched group) are predominantly found on the antennae. Several other components like 1,3-pentacosadiene and oxygenated aliphatic compounds occur in minor amounts on the cuticle. The reason for this polymorphism is discussed.

Animals↗
PubMed2004

Initial stages of low-temperature induction of cabbage cold shock protein gene csp5.

Some stages of low-temperature signal transduction causing appropriate cold stress response in plants are considered. The effects of Ca2+ chelators, Ca2+ channel blockers, and protein kinase inhibitors on protoplasts and plants of cabbage suggest that the initial stages of cold signal transduction are the change in membrane fluidity followed by the activation of calcium channels and elevation of Ca2+ influx into the cytoplasm. Increased concentration of Ca2+ in cytoplasm activates calcium-dependent protein kinase most likely participating in induction of transcription factors necessary for the expression of cold-regulated genes, in particular csp5. The protein kinase inhibitors staurosporine and wortmannin insignificantly repress the expression of csp5.

Brassica↗
PubMed2003

Proteolytic sensitivity of a recombinant phospholipase D from cabbage: identification of loop regions and conformational changes.

A recombinant phospholipase D from white cabbage (PLD2) composed of 812 amino acid residues was studied by site-directed mutagenesis and limited proteolysis to obtain first information on its tertiary structure. Limited proteolysis by thermolysin resulted in the formation of some large fragments of PLD2. From mass spectrometry and N-terminal sequencing of the peptides, the cleavage sites could be identified (1. Thr41-Ile42, 2. Asn323-Leu324 or Gly287-Leu288 and Ser319-Ile320 in case of the mutant L324S-PLD2). This suggested an exposed loop in the C2 domain of PLD2 and a large flexible region close to the N-terminal side of the first catalytic (HKD) motif. Calcium ions, the substrate 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and the competitive inhibitor 1,3-dipalmitoylglycero-2-phosphocholine influenced the proteolytic cleavage. Calcium ions exerted a destabilizing effect on the conformation of PLD2.

Amino Acid Sequence↗
PubMed2004

Behaviour of a recombinant cabbage (Brassica oleracea) phospholipase D immobilized on CNBr-activated and antibody supports.

Recombinant cabbage (Brassica oleracea) PLD2 (phospholipase D2) immobilized covalently on CNBr-activated Sepharose expressed low activity (approximately 10%), while that immobilized by binding on to anti-PLD2 IgG-Sepharose was more active (approximately 38%). Coupling of PLD2 to CNBr-activated Sepharose resulted in significant improvement in storage stability without affecting its thermostability, as compared with the soluble enzyme. Binding of PLD2 to the antibody support, however, rendered the enzyme remarkably labile to high temperatures and storage.

Animals↗
PubMed2005

Mannosamine supplementation extends the N-acetylglucosaminylation of recombinant human secreted alkaline phosphatase produced in Trichoplusia ni (cabbage looper) insect cell cultures.

A major limitation of the insect cell-baculovirus expression vector system is its poor capacity to perform complex glycosylation, since the glycoproteins produced are usually only of the high-mannose and paucimannose types. Nonetheless, recent evidence indicates that, under various conditions, some insect cell lines are capable of producing complex-type glycans. In the present study, we assessed the effects of supplementation with ManN (mannosamine) ManNAc (N-acetylmannosamine) and Cyt (cytidine) on the glycosylation of recombinant human secreted alkaline phosphatase produced by suspension cultures of Trichoplusia ni (cabbage looper) BTI-Tn5B1-4 cells. Addition of ManN in the range 5-20 mM resulted in a 10-fold increase of the terminal GlcNAc (N-acetylglucosamine) associated with the recombinant protein produced after baculovirus infection. Such an increase yielded a maximum of 12.5% hybrid glycans having terminal GlcNAc with respect to total N-linked glycans. In contrast, no changes in the glycan composition associated with recombinant human secreted alkaline phosphatase were observed on supplementation with up to 20 mM ManNAc or up to 1.5 mM Cyt. N-acetyl-beta-glucosaminidase activity in cell extracts was decreased on incubation with 20 mM ManN, but not with 20 mM ManNAc or 1.5 mM Cyt, indicating that the increased proportion of hybrid glycans obtained on the addition of ManN could be a result of N-acetyl-beta-glucosaminidase inhibition.

Alkaline Phosphatase↗
PubMed1967

The action of cabbage-leaf phospholipase D upon lysolecithin.

1. The action of the water-soluble phospholipase D from Savoy cabbage leaves upon an aqueous solution of lysolecithin has been studied. 2. Optimum conditions required the presence of Ca(2+) ions and pH5.8. 3. Equivalence was found between the amounts of lysolecithin hydrolysed and free choline released. The reaction was not accompanied by deacylation. 4. As the enzymic degradation proceeded, an opalescence developed, owing to the insolubility of the reaction product in the presence of Ca(2+) ions. 5. Evidence has been obtained indicating the heterogeneity of this reaction product.

Calcium↗
PubMed1967

The chemical nature of the products obtained by the action of cabbage-leaf phospholipase D on lysolecithin: the structure of lysolecithin.

1. Lysolecithin, prepared by the action of snake-venom phospholipase A on ovolecithin, when incubated with Savoy-cabbage phospholipase D, in the presence of Ca(2+) ions, gave two degradation products (designated A and B) in the form of their calcium salts. 2. These calcium salts were separated quantitatively by solvent fractionation and converted into the corresponding sodium salts. 3. Substance B proved to be a lysophosphatidic acid of conventional structure (1-monoacyl-l-3-glycerophosphoric acid). When the phosphate group was removed by means of prostatic acid phosphomonoesterase, a 1-monoglyceride was formed quantitatively. Alkaline hydrolysis gave the theoretical yield of l-3-glycerophosphate. 4. Substance A, on the other hand, had all the properties expected for a cyclic phosphate of a 1-monoglyceride. It was unaffected by phosphomonoesterase. On alkaline hydrolysis, the acyl group was removed and ring opening of the presumed cyclic phosphate group gave an approximately equimolar mixture of 2- and l-3-glycerophosphates. 5. The structures of substances A and B confirm lysolecithin as 1-monoacyl-l-3-glycerylphosphorylcholine.

Acid Phosphatase↗
PubMed1999

Cloning and expression of a novel juvenile hormone-metabolizing epoxide hydrolase during larval-pupal metamorphosis of the cabbage looper, Trichoplusia ni.

A full-length cDNA encoding for a microsomal juvenile hormone (JH)-metabolizing epoxide hydrolase (TmEH-1) was isolated from a cDNA library constructed from fat body of last stadium (wandering) cabbage loopers, Trichoplusia ni, at the exact developmental time of maximum JH epoxide hydrolase activity. TmEH-1 was 1887 base pairs in length with a 1389 base pair open reading frame encoding 463 amino acids. Amino acid sequence analysis showed that TmEH-1 was most similar to and contained the exact catalytic triad (Asp-226, Glu-403 and His-430) found in microsomal epoxide hydrolases. TmEH-1-specific message was present along with JH III epoxide hydrolase activity in fat body in feeding (days 1 and 2) and wandering (day 3) larvae with the peak in message level preceding the peak in JH epoxide hydrolase activity by 1 day. When TmEH-1 was expressed in baculovirus-infected Spodoptera frugiperda cells, a 46,000 molecular weight protein appeared on SDS-PAGE which corresponded to the predicted size coded by the TmEH-1 message and which was positively correlated with increases in JH III epoxide hydrolase activity above that of wild-type controls. In subcellular distribution studies, 58% of the juvenile hormone III epoxide hydrolase activity was in the insoluble fractions. Baculovirus expressed TmEH-1 demonstrated a higher specific activity for JH III as compared to the general EH substrates, cis- and trans-stilbene oxide. Southern blot analyses suggested that multiple epoxide hydrolase genes are present in T. ni.

Amino Acid Sequence↗
PubMed2003

Structural requirements for the perception of ambient temperature signals in homeothermic heat production of skunk cabbage (Symlocarpus foetidus).

The spadix of skunk cabbage, Symplocarpus foetidus, is capable of maintaining an internal temperature of around 20 degrees C even when the ambient temperature drops to around 0 degrees C. To determine the crucial structure that is required for detection of ambient temperature signals, detailed measurements of the temperatures of the spadix were made under field conditions. The spadix temperature was well regulated even when the spathe or the leaf of the plant was removed. Furthermore, maintenance of the temperature of the central stalk at either 10 or 20 degrees C had no effect on the thermoregulation when the ambient temperature increased from 10 to 25 degrees C or decreased from 20 to 8 degrees C. Therefore, it seemed that the heat production in the spadix required neither the spathe, the leaf, nor the central stalk for perception of the external temperature signals. Finally, analysis of sugar composition in xylem exudates showed that the concentrations of sucrose, glucose, and fructose, all of which are potential energy sources of thermogenesis, did not change significantly at different ambient temperatures. It is concluded that the spadix is a unique organ in which the perception of ambient temperature signals and heat production occurs in S. foetidus.

Journal Article↗
PubMed2003

Functionally distinct dopamine and octopamine transporters in the CNS of the cabbage looper moth.

A cDNA was cloned from the cabbage looper Trichoplusia ni based on similarity to other cloned dopamine transporters (DATs). The total nucleotide sequence is 3.8 kb in length and contains an open reading frame for a protein of 612 amino acids. The predicted moth DAT protein (TrnDAT) has greatest amino acid sequence identity with Drosophila melanogasterDAT (73%) and Caenorhabditis elegansDAT (51%). TrnDAT shares only 45% amino acid sequence identity with an octopamine transporter (TrnOAT) cloned recently from this moth. The functional properties of TrnDAT and TrnOAT were compared through transient heterologous expression in Sf9 cells. Both transporters have similar transport affinities for DA (Km 2.43 and 2.16 micro m, respectively). However, the competitive substrates octopamine and tyramine are more potent blockers of [3H]dopamine (DA) uptake by TrnOAT than by TrnDAT. D-Amphetamine is a strong inhibitor and l-norepinephrine a weak inhibitor of both transporters. TrnDAT-mediated DA uptake is approximately 100-fold more sensitive to selective blockers of vertebrate transporters of dopamine and norepinephrine, such as nisoxetine, nomifensine and dibenzazepine antidepressants, than TrnOAT-mediated DA uptake. TrnOAT is 10-fold less sensitive to cocaine than TrnDAT. None of the 15 monoamine uptake blockers tested was TrnOAT-selective. In situ hybridization shows that TrnDAT and TrnOAT transcripts are expressed by different sets of neurons in caterpillar brain and ventral nerve cord. These results show that the caterpillar CNS contains both a phenolamine transporter and a catecholamine transporter whereas in the three invertebrates whose genomes have been completely sequenced only a dopamine-selective transporter is found.

Amino Acid Sequence↗
PubMed2001

Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells.

Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1-233 or 234-850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing "nicked" pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment then exhibits cytotoxicity.

ADP Ribose Transferases↗
PubMed2001

Mono(ADP-ribosyl)ation of 2'-deoxyguanosine residue in DNA by an apoptosis-inducing protein, pierisin-1, from cabbage butterfly.

Pierisin-1 is a potent apoptosis-inducing protein derived from the cabbage butterfly, Pieris rapae. It has been shown that pierisin-1 has an A small middle dotB structure-function organization like cholera or diphtheria toxin, where the "A" domain (N-terminal) exhibits ADP-ribosyltransferase activity. The present studies were designed to identify the target molecule for ADP-ribosylation by pierisin-1 in the presence of beta-[adenylate-(32)P]NAD, and we found DNA as the acceptor, but not protein as is the case with other bacteria-derived ADP-ribosylating toxins. ADP-ribosylation of tRNAs from yeast was also catalyzed by pierisin-1, but the efficiency was around 110 of that for calf thymus DNA. Pierisin-1 efficiently catalyzed the ADP-ribosylation of double-stranded DNA containing dG small middle dotdC, but not dA small middle dotdT pairs. The ADP-ribose moiety of NAD was transferred to the amino group at N(2) of 2'-deoxyguanosine to yield N(2)-(alpha-ADP-ribos-1-yl)-2'-deoxyguanosine and its beta form, which were determined by several spectral analyses including (1)H- and (13)C-NMR and mass spectrometry. The chemical structures were also ascertained by the independent synthesis of N(2)-(D-ribos-1-yl)-2'-deoxyguanosine, which is the characteristic moiety of ADP-ribosylated dG. Using the (32)P-postlabeling method, ADP-ribosylated dG could be detected in DNA from pierisin-1-treated HeLa cells, in which apoptosis was easily induced. Thus, the targets for ADP-ribosylation by pierisin-1 were concluded to be 2'-deoxyguanosine residues in DNA. This finding may open a new field regarding the biological significance of ADP-ribosylation.

ADP Ribose Transferases↗
PubMed1998

Cloning and functional expression of a cDNA encoding a pheromone gland-specific acyl-CoA Delta11-desaturase of the cabbage looper moth, Trichoplusia ni.

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Delta9-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Delta9-desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni, a species in which all unsaturated pheromone products are produced via a Delta11Z-desaturation mechanism. The largest ORF of the approximately 1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Delta11Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Delta9-desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Delta9Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an approximately 1,250-nt PDesat-Tn Delta11Z mRNA that is consistent with the spatial and temporal distribution of Delta11-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Delta11Z resulted in complementation of the strain's fatty acid auxotrophy and the production of Delta11Z-unsaturated fatty acids.

Amino Acid Sequence↗
PubMed1999

Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: possible involvement of ADP-ribosylation in its activity.

We have previously reported that the cabbage butterfly, Pieris rapae, contains a 98-kDa protein, named pierisin, that induces apoptosis in a variety of human cancer cell lines. In the present study, sequencing and cloning of a cDNA encoding pierisin was accomplished. PCR-direct sequencing showed that the gene encodes an 850-amino acid protein with a calculated molecular weight of 98,081. An intact clone at the amino acid level encompassing the entire coding region was obtained by recombination of two independent clones, and the molecular mass of its in vitro expressed protein was about 100 kDa on SDS/PAGE, the same as that of purified native pierisin. The expressed protein induced apoptosis in human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, like the native protein, indicating functional activity. The deduced amino acid sequence of pierisin showed 32% homology with a 100-kDa mosquitocidal toxin from Bacillus sphaericus SSII-1. In addition, pierisin showed regional sequence similarities with ADP-ribosylating toxins, such as the A subunit of cholera toxin. A glutamic acid residue at the putative NAD-binding site, conserved in all ADP-ribosylating toxins, was also found in pierisin. Substitution of another amino acid for glutamic acid 165 resulted in a great decrease in cytotoxicity and induction of apoptosis. Moreover, inhibitors of ADP-ribosylating enzymes reduced pierisin-induced apoptosis. These results suggest that the apoptosis-inducing protein pierisin might possess ADP-ribosylation activity that leads to apoptosis of the cells.

ADP Ribose Transferases↗
PubMed2002

A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase.

A cDNA, PHCC-TPx, specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases was isolated from a Chinese cabbage cDNA library. PHCC-TPx encodes a preprotein of 232 amino acids containing a putative N-terminal chloroplast targeting sequence and three conserved Cys residues (Cys(107), Cys(136), and Cys(155)). The mature form of enzyme without the signal peptide was expressed in Escherichia coli, and the recombinant protein was found to utilize thioredoxin (Trx) but not GSH as an electron donor. In the presence of a Trx system, the protein efficiently reduces H(2)O(2) and organic hydroperoxides. Complementation analysis shows that overexpression of the PHCC-TPx restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the reducing agent of PHCC-TPx in vivo is not GSH but is Trx. Mutational analysis of the three Cys residues individually replaced with Ser shows that Cys(107) is the primary attacking site by peroxide, and oxidized Cys(107) reacts with Cys(155)-SH to make an intramolecular disulfide bond, which is reduced eventually by Trx. Tryptic peptide analysis by matrix-assisted laser desorption and ionization time of flight mass spectrometry shows that Cys(155) can form a disulfide bond with either Cys(107) or Cys(136).

Amino Acid Sequence↗
PubMed2003

Identification of glycosphingolipid receptors for pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly.

Pierisin-1, a cytotoxic protein found naturally in the cabbage butterfly, induces apoptosis of mammalian cells. Our recent studies suggest that pierisin-1 consists of an N-terminal ADP-ribosyltransferase domain, and a C-terminal region that binds to receptors on the surfaces of target cells and incorporates the protein into cells. The present study was undertaken to identify receptors for pierisin-1. The cross-linking and cloning experiments suggested that the proteins on cell membrane had no binding ability to pierisin-1. Inhibitory assays of fractionated lipids from human cervical carcinoma HeLa cells, which are highly sensitive to pierisin-1, indicated neutral glycosphingolipids on the cell surface to show receptor activity. Inhibitory assays and TLC immunostaining using anti-pierisin-1 antibodies demonstrated two neutral glycosphingolipids as active components. Analysis of their structures with glycosphingolipid-specific antibodies and negative secondary ion mass spectrometry identified them as globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4). The receptor activities of Gb3 and Gb4 for pierisin-1 were also confirmed with these authentic compounds. Pierisin-1-insensitive mouse melanoma MEB4 cells were found to lack pierisin-1 receptors, including Gb3 and Gb4, but pretreatment of the cells with glycosphingolipid Gb3 or Gb4 enhanced their sensitivity to pierisin-1. Thus, Gb3 and Gb4 were proven to serve as pierisin-1 receptors. The C-terminal region of pierisin-1 consists of possible lectin domains of a ricin B-chain, containing QXW sequences, which are essential for its structural organization. Alteration of QXW by site-directed mutagenesis caused marked reduction of pierisin-1 cytotoxicity. Thus, our results suggest that pierisin-1 binds to Gb3 and Gb4 receptors at the C-terminal region, in a manner similar to ricin, and then exhibits cytotoxicity after incorporation into the cell.

ADP Ribose Transferases↗
PubMed2002

Effect of cabbage cultivars with varying levels of resistance to aphids on the performance of the parasitoid, Aphidius colemani (Hymenoptera: Braconidae).

The effect of aphid resistance in plant cultivars on parasitism of Myzus persicae (Sulzer) and Brevicoryne brassicae (Linnaeus) by Aphidius colemani Viereck was investigated under laboratory conditions using three cultivars of common cabbage, Brassica oleracea var. capitata. Significantly greater aphid populations were found on cv. Derby Day (green-leaved, susceptible to both aphid species), regardless of the presence or absence of parasitoids, compared with cv. Minicole (green-leaved, partially resistant with antibiosis factors for B. brassicae) or cv. Ruby Ball (red-leaved, partially resistant with antixenosis factors for B. brassicae). Minicole had the greatest proportion of parasitized aphids and Derby Day the least. A significantly lower percentage of emerged parasitoids were recorded on Minicole for both aphid species compared with Derby Day and Ruby Ball. The implication of the results are discussed in relation to the integrated control of aphids.

Animals↗
PubMed2002

Tritrophic interactions between cabbage cultivars with different resistance and fertilizer levels, cruciferous aphids and parasitoids under field conditions.

Tritrophic interactions involving cabbage Brassica oleracea var. capitata cultivars +/- fertilizer, Brevicoryne brassicae (Linnaeus) and Myzus persicae (Sulzer), and the parasitoids Diaeretiella rapae (M'cIntosh) and Aphidius sp. were conducted in 1998 and 1999. Brevicorne brassicae was the dominant aphid species on all cultivars +/- fertilizer, except for some treatments in late season 1998. Ruby Ball (red-leaved with antixenosis factors for B. brassicae alates) +/- fertilizer was consistently less colonized by aphids in early stages of plant growth, although only significantly so compared with Derby Day (green-leaved, susceptible to aphids) without fertilizer for B. brassicae and Minicole (green-leaved with antibiosis factors for B. brassicae) with fertilizer for M. persicae. In early 1999, only B. brassicae was present and no significant differences between cultivars were seen. In the mid to late season 1998, the highest aphid infestations were usually found on Derby Day, although only significantly so for B. brassicae, in some treatments. In 1999, higher aphid infestations were observed on Derby Day in mid to late season and some significant differences were found for M. persicae as well as for B. brassicae. In both years, Ruby Ball had the greatest mummy:aphid ratios early season, with no consistent difference between the other cultivars. Later in the season, mummy:aphid ratios were generally highest on Minicole. Parasitism differed in seasonal occurrence and relative abundance. Diaeretiella rapae mummies were found earlier than Aphidius sp. There was evidence of a beneficial interaction between the degree of plant resistance and biological control in early to mid season.

Animals↗