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High-performance liquid-phase separation of glycosides. III. Determination of total glucosinolates in cabbage and rapeseed by capillary electrophoresis via the enzymatically released glucose.

A selective and sensitive method for the determination of total glucosinolates (GSs) in plant extracts by capillary electrophoresis-laser-induced fluorescence (LIF) detection was developed. It was based on the enzymatically released glucose from glucosinolates in the presence of the hydrolyzing enzyme myrosinase. The released glucose was converted to gluconic acid (GA) by the action of glucose oxidase. The resulting GA was then labeled selectively with the fluorescent tag 7-aminonaphthalene-1, 3-disulfonic acid (ANDSA). The peak area resulting from the GA-ANDSA derived from free and bound glucose was subtracted from the peak area of the GA-ANDSA resulting from the free glucose in the sample. This gave the total glucosinolates in the sample. The peak areas were normalized to the internal standard, N-acetylneuraminic acid derivatized with ANDSA. The method was validated using four different plant extracts, white cabbage leaves, rapeseed leaves, rapeseed roots, and rapeseed seeds. Furthermore, a capillary electrophoresis-UV detection method for profiling GS in plant extracts was developed. In addition to providing a fingerprint of the glucosinolates in plant extracts, the method allowed the experimenter to rapidly check the various steps involved in the extraction and sample cleanup.

2-Naphthylamine↗

Screening for Bacillus thuringiensis crystal proteins active against the cabbage looper, Trichoplusia ni.

Toxicity tests were performed to find among Cry1 and Cry2 Bacillus thuringiensis crystal proteins those with high activity against the cabbage looper. Tests were performed with neonate larvae on surface-contaminated artificial diet. The crystal proteins found to be toxic were, from higher to lower toxicity: Cry1Ac, Cry1Ab, Cry1C, Cry2Aa, Cry1J, and Cry1F (LC50 of 1.14.1, 3.4-4.4, 12, 34, 87, and 250 ng/cm2, respectively). Cry1B, Cry1D, and Cry1E can be considered nontoxic (LC50 higher than 2500 ng/cm2). Cry1Aa was moderately toxic to nontoxic, depending on the source (LC50 of 420 ng/cm2 from PGS and 8100 ng/cm2 from Ecogen). In vitro binding assays with trypsin-activated 125I-labeled Cry1Aa, Cry1Ab, and Cry1Ac crystal proteins and brush border membrane vesicles from midgut larvae showed a direct correlation between toxicity and binding affinity. Heterologous competition experiments indicated that Cry1Aa and Cry1F bind, though only at very high concentrations, to the Cry1Ab/Cry1Ac shared high-affinity binding site.

Animals↗

Surface morphology of peritrophic membrane formation in the cabbage looper, Trichoplusia ni.

The development of the peritrophic membrane in the cabbage looper, Trichoplusia ni, was investigated by scanning electron microscopy. The development of this membrane is characterized by a series of events suggested by the observations to be (1) secretion of material among and above the microvilli of the midgut epithelial cells, (2) maturation of this material into a randomly cross-linked fibrous matrix, and (3) aggregation of amorphous materials in and within the matrix. The membrane, possessing small discontinuities, remains intact in the midgut, but shows gross damage by the time it is passed from the insect, surrounding the feces.

Animals↗

An insect model for assessing mercury toxicity: effect of mercury on antioxidant enzyme activities of the housefly (Musca domestica) and the cabbage looper moth (Trichoplusia ni).

The effect of mercury as Hg2Cl2 and HgCl2 on the antioxidant enzyme levels and its toxicity was investigated in an insect model comprised of adult females of the common housefly, Musca domestica, and fourth-instar larvae of the cabbage looper moth, Trichoplusia ni. HgCl2 was found to be more toxic than Hg2Cl2 to both M. domestica and T. ni. The LC50s for M. domestica were 1.17% and 0.38% w/v concentration for Hg2Cl2 and HgCl2, respectively. For the more tolerant T. ni, the LC50S were 5.15% for Hg2Cl2 and 0.96% w/w concentration for HgCl2. The minimally acute LC5 dose of both oxidation states of Hg was approximately 0.005% for both insects (w/v for M. domestica and w/w for T. ni). At the LC5, both forms of Hg significantly induced the activity of superoxide dismutase in both insect species. Catalase was induced by both Hg2Cl2 and HgCl2 in M. domestica but was only induced by HgCl2 in T. ni. Glutathione-S-transferase, its peroxidase activity, and glutathione reductase activities were also significantly altered in most cases by Hg in both insects although the pattern of alternation was different between the two insects. It is evident that mercury induces oxidative stress in insects as it does in vertebrates. Our findings suggest that insects may serve as a valuable, non-mammalian model species to assess Hg-induced oxidative stress as a component of environmental toxicity.

Animals↗

Fine structure of the ocellus of the cabbage looper moth (Trichoplusia ni).

The dorsal ocelli of adult cabbage looper moths were studied by light microscopy, and scanning and transmission electron microscopy. Each ocellus has a cuticular lens located on the distal end of a cuticular cone which encapsulates the receptor cells. There are two distinct types of receptor cells in the ocellus. Seventy large receptor cells from plate-like rhabdoms with several adjacent cells to produce a rhabdom network in the ocellus. Proximally ninety small receptor cells which have a disorganized microvillar rhabdomere are located at the base of the rhabdoms formed by the large cells. Extensive areas of gap junctions which occur between the rhabdoms and the membranes of large and small cells suggest that the cells may be electrically coupled to one another. Axons from both large and small receptor cells leave the base of the ocellus and extend to the protocerebrum to synapse with second order neurons.

Animals↗

[Preparation and characterization of low protein, cell structural material. II. Materials with cellular structure from carrot and white cabbage].

The molecular parameters and functional properties of low-protein materials with a cellular structure are determined by both the conditions of preparation and the kind of raw material used. Compared to alcohol-insoluble substance, the conditions of preparation were modified. When an alkaline extraction and a favorable combination of processing steps is used, materials with special low protein and high pectin contents result. In addition, such functional properties as water binding capacity and cation exchange capacity of the materials are improved. After having prepared various materials with a cellular structure from apple, carrot, and white cabbage, it was found that the amount of solvent needed and the yield, as well as the molecular parameters and the functional properties were also determined by the kind of raw materials used. For instance, material with a cellular structure from apple is low in protein and high in water binding capacity. Whereas, materials with a cellular structure from carrot, which contains a deesterified pectin component, is characterized by a good yield, a high pectin content, and an excess of 1.5 mmol/g of free carboxyl groups.

Brassica↗

Growth of nuclear polyhedrosis virus in larvae of the cabbage moth, Mamestra brassicae L.

Growth of nuclear polyhedrosis virus (NPV) in 5 larval instars of cabbage moth, Mamestra brassicae, has been quantified using 2 methods. Numbers of polyhedra were estimated by light microscope counts while concentrations of virus protein antigen were estimated using ELISA. Virus growth was rapid initially but slowed during its later stages, although ELISA protein concentrations decreased once a peak had been reached. There was a linear correlation between polyhedral counts and virus protein during the initial growth phase. Maximum polyhedral production ranged from 2 x 107 (first instar) to 3.4 x 109 (fifth instar) and could be correlated directly to increasing larval weight. Using ELisa, virus antigen was detectable at least 24 hours before polyhedra were observed under the light microscope. Productivity ratios ranged from 83,500 in the first instar to 1352 in the fifth instar.

Animals↗

2-Phenylethanol, a presumed sexual stimulant produced by the male cabbage looper moth, Trichoplusia ni.

A sex pheromone produced by male cabbage looper moths, Trichoplusia ni (Hübner), has been isolated from the genital scent brushes and identified as 2-phenylethanol. It is shown conclusively to elicit specific behavioural responses in the female (such as wing vibration and abdominal elevation), as determined by a novel behavioural laboratory bioassay. This is taken as further evidence that the male pheromone of T. ni acts as a sexual stimulant (aphrodisiac) prior to mating. 2-Phenylethanol represents the first identification of a genital scent brush pheromone in the family Noctuidae, and of a male pheromone in the subfamily Plusiinae.

Action Potentials↗

Purification and characterization of histidinol dehydrogenase from cabbage.

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.

Alcohol Oxidoreductases↗

Conversion of radiolabelled ethanolamine plasmalogen into the dimethylethanolamine and choline analogue via transphosphatidylation by phospholipase D from cabbage.

Starting from biosynthetically prepared ethanolamine plasmalogen 14C-labelled in the O-alkenyl moiety, choline and dimethylethanolamine plasmalogen were prepared by transphosphatidylation utilizing phospholipase D from cabbage. Investigation of the time course of the reaction showed that transphosphatidylation was simultaneously accompanied by hydrolysis of both the substrate and the desired product, resulting in a maximum of product yield after 1-3 h under the reaction conditions investigated. Optimal reaction conditions gave yields of 40% and 62% (of total radioactivity) respectively for the purified choline and dimethylethanolamine derivatives.

Animals↗

A mutagen precursor in Chinese cabbage, indole-3-acetonitrile, which becomes mutagenic on nitrite treatment.

After treatment with nitrite, Chinese cabbage showed direct-acting mutagenicity on Salmonella typhimurium TA100 inducing 3100 revertants per g. One of the mutagen precursors that became mutagenic after nitrite treatment was isolated, and identified as indole-3-acetonitrile. After treatment with nitrite, 1 mg of indole-3-acetonitrile induced 17 400 revertants of TA100 and 21 000 revertants of TA98 without S9 mix.

Biotransformation↗

In vivo mass production in the cabbage moth (Mamestra brassicae) of a heterologous (Panolis) and a homologous (Mamestra) nuclear polyhedrosis virus.

In preparation for field trials, a nuclear polyhedrosis virus (NPV) of the pine beauty moth, Panolis flammea, was mass produced in vivo in an alternative (heterologous) host, the cabbage moth, Mamestra brassicae. Simultaneously, Mamestra NPV was also produced in M. brassicae. This homologous NPV/host system was a consistently more efficient production system than the heterologous one. Evidence is presented that increased humidity may improve levels of infection and, therefore, yield achieved. Full purification of the resultant product through a sucrose gradient resulted in a loss of virus of about 30%.

Animals↗

Chemometric studies of the effect of toxic metals on plants: the use of response surface methodology to investigate the influence of Tl, Cd and Ag on the growth of cabbage seedlings.

The growth of cabbage seedlings at five concentrations of Cd, Tl and Ag was monitored by measuring their wet weights, root and shoot lengths after four week's growth in a controlled environment. Toxicity curves were constructed using a three factor central composite experimental design. The interaction and competition between any two metals were studied by displaying response surfaces. Ag was observed to be the most toxic, while Tl and Cd, although toxic, exhibited fairly similar effects. Analysis of variance was used to test for the significance of each of the computed parameters.

Journal Article↗

Effects of dietary cabbage, Brussels sprouts, Illicium verum, Schizandra chinensis and alfalfa on the benzo[alpha]pyrene metabolic system in mouse liver.

Male C57B16 mice were fed on diets containing either 20% cabbage, 20% Brussels sprouts, 20% alfalfa, 5% Schizandra chinensis or 5% Illicium verum (two Chinese medicinal herbs) or on a chow or purified basal diet for 14 days after a 1-wk equilibration period on the basal diet. Liver microsomal fractions were assayed for cytochrome P-450 content, aryl hydrocarbon hydroxylase (AHH) and epoxide hydrolase (EH). Liver microsome-mediated benzo[a]pyrene (BP) metabolism (with and without an EH inhibitor, 1,2-epoxy-3,3,3-trichloropropane) was analysed by HPLC. Liver weights of the animals fed on Brussels sprouts and I. verum were significantly increased compared with those of the animals fed on basal diets. S. chinensis induced a 3-fold increase in cytochrome P-450 (P less than 0.05). Although P-450 induction in the other groups was as high as 1.8-fold (for chow), statistical significance was not established. Chow induced AHH activity 2.2-fold (P less than 0.05), while S. chinensis and alfalfa induced 1.6-fold and 1.7-fold increases, respectively, in AHH activity, although neither increase was statistically significant. EH was stimulated significantly in the following order: I. verum (2.1-fold) greater than chow (1.7-fold) greater than S. chinensis (1.6-fold) greater than Brussels sprouts (1.4-fold). Total levels of BP metabolism and phenol II (primarily 3-hydroxybenzo[a]pyrene) formation were closely associated for each dietary treatment. Total BP metabolism was significantly increased (2.1-fold) in the chow-fed group and increased 1.6-fold in the S. chinensis group (P greater than 0.05). No increase was seen with the other diets. Phenol II formation relative to total metabolites was significantly increased for the S. chinensis and I. verum groups compared to the basal group. Diet-related variations in phenol production relative to total metabolism were eliminated by addition of the EH inhibitor to the incubation media.

Animal Feed↗

Improved purification and biochemical characterization of phospholipase D from cabbage.

Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was purified from cabbage leaves. The two step purification procedure involved hydrophobic chromatography on Octyl-Sepharose followed by a Mono-Q/FPLC-column with a total yield of 23% and a purification factor of 1000. A zymographic assay was used to detection of PL D activities at various stages of purification under non denaturing PAGE. The molecular mass was determined to be 90 kDa using the SDS/PAGE method, and 90,200 Da as calculated from the amino acid analysis. The isoelectric point of the enzyme is acidic (pI = 4.7). The amino-acid composition and 29 residues of the NH2-terminal amino-acid sequence were determined.

Amino Acid Sequence↗

Mono(ADP-ribosyl)ation of the N2 amino groups of guanine residues in DNA by pierisin-2, from the cabbage butterfly, Pieris brassicae.

Pierisin-2 is a cytotoxic and apoptosis-inducing protein present in Pieris brassicae with a 91% homology in the deduced amino acid sequences to pierisin-1 from Pieris rapae. We earlier showed pierisin-1 to catalyze mono(ADP-ribosyl)ation of 2'-deoxyguanosine (dG) in DNA to form N2-(ADP-ribos-1-yl)-2'-deoxyguanosine, this DNA modification appearing linked to its cytotoxicity and ability to induce apoptosis in mammalian cell lines. In this paper, we documented evidence that pierisin-2 also catalyzed ADP-ribosylation of dG in DNA to give the same reaction product as demonstrated for pierisin-1, with similar efficiency. With oligonucleotides as substrates, ADP-ribosylation by pierisin-2 was suggested to occur by one-side attack of the carbon atom at 1 position of the ribose moiety in NAD toward N2 of dG. The presence of a unique ADP-ribosylation toxin targeting dG in DNA in two distinct species in a Pieris genus could be a quite important finding to better understand biological functions of pierisin-1 and -2 in Pieris butterflies and the generic evolution of these cabbage butterflies.

ADP Ribose Transferases↗

Functional analysis of skunk cabbage SfUCPB, a unique uncoupling protein lacking the fifth transmembrane domain, in yeast cells.

Skunk cabbage, Symplocarpus foetidus, expresses two uncoupling proteins (UCPs), termed SfUCPA and SfUCPB, in the thermogenic organ spadix. SfUCPB exhibits unique structural features characterized by the absence of the putative fifth transmembrane domain (TM5) observed in SfUCPA, which is structurally similar to UCP1, and is abundantly expressed in the thermogenic spadix. Here, we conducted a series of comparative analyses of UCPs with six transmembrane domains, SfUCPA and rat UCP1, and TM5-deficient SfUCPB, using a heterologous yeast expression system. All UCPs were successfully expressed and targeted to the mitochondria, although the expression level of SfUCPB protein was approximately 10% of rat UCP1. The growth rate, mitochondrial membrane potential, and ATP content were significantly lower in cells expressing SfUCPB than in those expressing rat UCP1 and SfUCPA. These results suggest that SfUCPB, a novel TM5-deficient UCP, acts as an uncoupling protein in yeast cells.

Adenosine Triphosphate↗