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PubMed2006

Characterization and cDNA cloning of hinnavin II, a cecropin family antibacterial peptide from the cabbage butterfly, Artogeia rapae.

Hinnavins, together with lysozymes, are the main types of antibacterial peptides/proteins previously isolated from the larval haemolymph of the cabbage butterfly, Artogeia rapae as part of the humoral immune response to a bacterial invasion. One of these antibacterial peptides, named hinnavin II, was purified and characterized after cDNA cloning. The purified hinnavin II was more active against Gram negative than against Gram positive bacteria. Hinnavin II also showed a powerful synergistic effect on the inhibition of bacterial growth with purified lysozyme. The cDNA has a total length of 186 bp with a 114 coding region. The deduced protein sequence contains 38 amino acids with a coding capacity of 4142.8 Da. The result of a multiple sequence alignment and phylogenetic analysis with Clustal W indicated that mature hinnavin II showed an approximately 78.9% amino acid sequence identity with cecropin A and originated from a group containing mostly lepidopteran cecropins.

Amino Acid Sequence↗
PubMed2006

Influence of lead acetate on soil microbial biomass and community structure in two different soils with the growth of Chinese cabbage (Brassica chinensis).

A greenhouse pot experiment was conducted to evaluate the impact of different concentrations of lead acetate on soil microbial biomass and community structure during growth of Chinese cabbage (Brassica chinensis) in two different soils. The field soils were used for a small pot, short-term 60-day growth chamber study. The soils were amended with different Pb concentrations, ranging from 0 to 900mgkg(-1) soil. The experimental design was a 2 soilx2 vegetation/non-vegetationx6 treatments (Pb)x3 replicate factorial experiment. At 60 days the study was terminated and soils were analyzed for microbial parameters, namely, microbial biomass, basal respiration and PLFAs. The results indicated that the application of Pb at lower concentrations (100 and 300mgkg(-1)) as lead acetate resulted in a slight increase in soil microbial biomass, whereas Pb concentrations >500mgkg(-1) caused an immediate gradual significant decline in biomass. However, the degree of impact on soil microbial biomass and basal respiration by Pb was related to management (plant vegetation) or the contents of clay and organic matter in soils. The profiles of 21 phospholipid fatty acids (PLFAs) were used to assess whether observed changes in functional microbial parameters were accompanied by changes in the composition of the microbial communities after Pb application at 0, 300 and 900mg Pbkg(-1) soil. The results of principal component analyses (PCA) indicated that there were significant increases in fungi biomarkers of 18:3omega6c, 18:1omega9c and a decrease in cy17:0, which is an indicator of gram-negative bacteria for the high levels of Pb treatments In a word, soil microbial biomass and community structure, therefore, may be sensitive indicators reflecting environmental stress in soil-Pb-plant system. However, further studies will be needed to better understand how these changes in microbial community structure might actually impact soil microbial community function.

Biomass↗
PubMed2005

Molecular characterization of four midgut aminopeptidase N isozymes from the cabbage looper, Trichoplusia ni.

Four aminopeptidase N (APN) isoforms, TnAPN1, TnAPN2, TnAPN3 and TnAPN4, were identified from the cabbage looper, Trichoplusia ni, by cDNA cloning. The deduced amino acid sequences of the four APNs indicate that TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are synthesized as pre-proteins of 110, 106, 114 and 108 kDa, respectively. Sequence features of the T. ni APNs include the presence of a signal peptide at their N-termini and a prepeptide at the C-termini for the GPI anchor, the zinc binding/gluzincin motif HEX2HX18E, the gluzincin aminopeptidase motif GAMENWG and the presence of glycosylation sites. After removal of the signal peptide and the C-terminal prepeptide, the predicted molecular weights of TnAPN1, TnAPN2, TnAPN3 and TnAPN4 are 106, 102, 110 and 104 kDa, respectively. Enzymatic activity assays of various larval tissues showed that aminopeptidase activities were mainly localized in the midgut and the specific enzyme activity per mg of midgut tissue proteins was constant in T. ni larvae regardless of the composition of dietary proteins and amino acids. Both enzyme activity assays and RT-PCR analyses for the expression of the APN genes in T. ni larval tissues demonstrated that APN genes were expressed in Malphigian tubules in addition to the midgut, which was the first observation that APNs were also synthesized in insect Malphigian tubules. The finding of APN gene expression and enzyme activity in the Malphigian tubules indicated the biochemical and functional similarity of the insect Malphigian tubules to the mammalian counterpart, the kidney, in which APNs are known to play important functions.

Amino Acid Sequence↗
PubMed2005

A novel chitin-binding protein identified from the peritrophic membrane of the cabbage looper, Trichoplusia ni.

A novel midgut peritrophic membrane (PM) protein, TnPM-P42, was identified from the cabbage looper, Trichoplusia ni. TnPM-P42 was shown as a 42kDa protein by SDS-PAGE analysis and appeared to be associated with the PM throughout its entire length. In T. ni larvae, the midgut is the only tissue where TnPM-P42 could be detected during the feeding period of the larvae. TnPM-P42 has chitin-binding activity and is strongly associated with the PM, which is similar to the currently known peritrophin type PM proteins. However, TnPM-P42 represents a unique family of proteins distinctly different from the peritrophin type PM proteins in its sequence characteristics. TnPM-P42 does not contain the peritrophin domain which is present in all the currently known PM proteins, but instead has a chitin deacetylase-like domain. Sequence similarity search of the GenBank database did not result in identification of any known proteins with a significant overall sequence similarity to the TnPM-P42. However, expressed sequence tags (ESTs) from various arthropods were identified to code for proteins with high sequence similarities to TnPM-P42, indicating the presence of TnPM-P42 homologs in other arthropods. Consistent with the identification of various ESTs from arthropods, Western blot analysis demonstrated the presence of a TnPM-P42-like protein in the PMs from Heliothis virescens and Helicoverpa zea larvae. The sequence characteristics of TnPM-P42 indicate that TnPM-P42 represents a novel family of insect proteins. However, its biochemical and physiological functions require further investigation.

Amino Acid Sequence↗
PubMed2004

Effect of common Brassica vegetables (Brussels sprouts and red cabbage) on the development of preneoplastic lesions induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in liver and colon of Fischer 344 rats.

Aim of the present study was the investigation of effects of juices from commonly consumed Brassica vegetables (two cultivars of Brussels sprouts and two cultivars of red cabbage) on formation and development of preneoplastic lesions in colons (aberrant crypt foci, ACF) and livers (glutathione-S-transferase placental form, GST-P+) in male F344 rats. The foci were induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a widespread carcinogenic heterocyclic aromatic amine which is found in fried meats. Recently, we reported on pronounced protective effects in the two-organ foci model when the vegetable juices were given during the carcinogen treatment but several findings by other groups indicated that breakdown products of glucosinolates contained in Brassica vegetables cause tumour promotion in various organs of laboratory rodents. In the present study, the animals received the juices in the drinking water (5%) over a period of 20 days after treatment with IQ (100 mg/kg bw on 10 alternate days). To increase the foci yield (which facilitates the detection of modifying effects), the animals were fed with a modified (high fat, fibre free) AIN-76 diet. With exception of the sprout variety "Cyrus", all juices lowered the number of GST-P+ foci as well as the foci area in the liver, but none of these effects was statistically significant. In the colon, none of the juices had an impact on crypt multiplicity (number of crypts/focus), whereas the number of ACF was decreased; only with the sprout variety Maximus the protective effect was significant (reduction 49%). The present findings show that administration of vegetable juices to the animals after the carcinogen does not increase the number and size of IQ-induced preneoplastic lesions in liver and colon.

Animals↗
PubMed2005

Sulfur, chromium, and selenium accumulated in Chinese cabbage under direct covers.

Currently, pollution of our agricultural soils and waters is increasing and is often associated with many human health ailments. Soils contaminated with low levels of heavy metals and other trace elements are frequently used for growing vegetable crops and in such a situation, these toxic contaminants often accumulate in the edible portions of these agricultural plants and thereby enter the human food chain. In 3 consecutive years of field experiments (1994-1996), two different crop-covering treatments--T (50 microm perforated polyethylene), and T2 (17 gm(-2) non-woven polypropylene)--were used to modify the environmental conditions for the growth of Chinese cabbage 'Nagaoka 50' [Brassica rapa L. (Pekinensis group)]. Open-air plots (T(0)) were used as controls. Analytical determinations of chromium (Cr), selenium (Se), two forms of sulfur (total-S and sulfate-S), and amino acids (Isoleucine, leucine, lysine, methionine, serine, threonine, and valine) were performed utilizing plant shoots for analysis. The T1 and T2 treated plants contained concentrations of lysine, methionine, serine, and threonine higher than in T(0). Under T1, the extent of Cr and Se removal in the field was more favorable. Direct covers could be used in contaminated agricultural zones for a variety of plant species, not just for use with those plants previously reported to be efficient at bio-accumulating toxic elements because the thermal effect created by the covers favors phytoextraction processes. However, it is clear that the accumulation of these toxic substances in the plants (Cr) would deem the plant material unsuitable for human consumption and use as animal fodder.

Amino Acids↗
PubMed2003

Physiological and biochemical changes in summer and winter diapause and non-diapause pupae of the cabbage armyworm, Mamestra brassicae L. during long-term cold acclimation.

Previous studies have demonstrated that cold hardiness in the cabbage armyworm Mamestra brassicae is more developed in winter diapause pupae (WD) than in summer diapause pupae (SD) after short-term cold acclimation. In this study, we investigated specific physiological and biochemical differences between diapause and non-diapause pupae (ND) of M. brassicae after acclimation at 5 degrees C for up to 4 months. The period required for adult emergence in ND pupae at 20 degrees C was about 20.2 d and independent of the months acclimated. However, SD and WD pupae reached this level only after 1- and 4-month acclimation, respectively. Levels of respiration (O(2) consumption and CO(2) release) in ND(0) (the subscript indicates the months of acclimation at 5 degrees C) was eightfold higher than in WD(0) and SD(0), decreased sharply in ND(1) and reached diapause pupae (WD and SD) levels in ND(2). Maximal cold hardiness developed during cold acclimation was found in WD(3), SD(2) and ND(2) pupae. During cold acclimation, glycogen content remained at higher levels in WD and SD than in ND. Lipid content in ND decreased but remained high in SD and WD. These results suggest that diapause pupae show large amounts of metabolic reserves in the form of glycogen and lipid during diapause development. To our knowledge, this is the first report to compare the physiological and biochemical differences among summer and winter diapause pupae and non-diapause pupae in the same insect species during long-term cold acclimation.

Acclimatization↗
PubMed2005

Molecular cloning and stress-dependent regulation of potassium channel gene in Chinese cabbage (Brassica rapa ssp. Pekinensis).

Potassium channels are important for many physiological functions in plants, one of which is to regulate plant adaption to stress conditions. In this study, KCT2, the gene encoding a membrane-bound protein potassium channel (GenBank accession number: ), was isolated from Chinese cabbage (Brassica rapa ssp. Pekinensis) by RACE-PCR technique. Bioinformatics methods were performed for the gene structure and molecular similarity analysis. The KCT2 expression patterns under various stress conditions were studied by semi-quantitative RT-PCR. DNA gel blot was used to analyze genomic organization. The putative KCT2 was found to contain five membrane-spanning segments, a pore-forming domain (P-domain) between the last two transmembrane spans, a TxxTxGYGD motif in the P-domain and a putative cyclic nucleotide-binding-like domain within a long C-terminal region. KCT2 is closest to KAT2 in Arabidopsis. KCT2 could be a one-copy gene with different isoforms or belong to a small gene family with four or five members. KCT2 was expressed more strongly in leaves than in shoots and roots. KCT2 transcription products were up-regulated by a 4-h-incubation in abscisic acid (ABA) and various stress treatment including cold stress (4 degrees C) for 24 h, drought stress for 1h, and salt stress for 12 h. KCT2 transcription was not affected by anoxia stress for 8h and was down-regulated with cold stress for 48 h. KCT2 was cloned for the first time from the genus Brassica. Expression analysis indicated that in the early stage of plant adaption to stress conditions KCT2 is up-regulated, which results in a stimulation of potassium transport.

Amino Acid Motifs↗
PubMed2005

Involvement of nucleotide excision repair (NER) system in repair of mono ADP-ribosylated dG adducts produced by pierisin-1, a cytotoxic protein from cabbage butterfly.

Pierisin-1, a cytotoxic protein from the cabbage butterfly (Pieris rapae), induces apoptosis in mammalian cell lines. Binding of its C-terminal region to glycosphingolipid Gb3 and Gb4 receptors on cell membrane is necessary for incorporation into cells, while the N-terminal polypeptide catalyzes transfer of the ADP-ribose moiety of NAD at N2 of dG in DNA. Resulting DNA adducts cause mutation if they are present at low levels. If the DNA damage is more severe, the cells undergo apoptosis. In the present study, we examined the repair system for ADP-ribosylated dG adducts using nucleotide excision repair (NER) mutants of Chinese hamster ovary (CHO) cell lines. Pierisin-1 showed cytotoxic effects in all cases: IC50 values of them were; 650 ng/ml for AA8 (wild), 230 ng/ml for UV5, 190 ng/ml for UV20, 260 ng/ml for UV41, and 240 ng/ml for UV135. Thus, wild-type AA8 proved most resistant to pierisin-1-induced cytotoxicity. When these CHO cell lines were treated with pierisin-1, the adduct levels of ADP-ribosylated dG increased to 2.5-4.8/10(5) nucleotides time-dependently in all cell lines at 12 h. After removal of pierisin-1, the adduct levels remained constant or increased to 4-14/10(5) nucleotides in all NER mutant cells (UV5, UV20, UV41, UV135), while those rapidly decreased to 0.27/10(5) nucleotides in the repair proficient AA8 cells for 24 h. From these results, it is suggested that the NER system is involved in the repair of ADP-ribosylated dG adducts in DNA.

ADP Ribose Transferases↗
PubMed2000

Hydrolytic and transphosphatidylation activities of phospholipase D from Savoy cabbage towards lysophosphatidylcholine.

The hydrolysis and transphosphatidylation of lysophosphatidylcholine (LPC), with a partially purified preparation of phospholipase D (PL D) from Savoy cabbage, was investigated. These reactions were about 20 times slower than the hydrolysis of phosphatidylcholine (PC) in a micellar system. For the transfer reaction, 2 M glycerol was included in the media, which suppressed the hydrolytic reaction. Both reactions presented similar V(max) values, suggesting that the formation of the phosphatidyl-enzyme intermediate is the rate-limiting step. The enzyme had an absolute requirement for Ca(2+), and the optimum concentration was approximately 40 mM CaCl(2). K(Ca)(app) was calculated to be 8.6+/-0.74 mM for the hydrolytic and 10+/-0.97 mM for the transphosphatidylation reaction. Both activities reached a maximum at pH 5.5, independent of Ca(2+) concentration. Kinetic studies showed that the Km(app) for the glycerol in the transphosphatidylation reaction is 388+/-37 mM. Km(app) for the lysophosphatidylcholine depended on Ca(2+) concentration and fell between 1 and 3 mM at CaCl(2) concentrations from 4 to 40 mM. SDS, TX-100, and CTAB did not activate the enzyme as reported for phosphatidylcholine hydrolysis; on the contrary, reaction rates decreased at detergent concentrations at or above that of lysophosphatidylcholine.

Brassica↗
PubMed1997

Role of dopamine at the onset of pupal diapause in the cabbage armyworm Mamestra brassicae.

Experiments were conducted to examine the relationship between an onset of diapause and dopamine (DA) content in the cabbage armyworm Mamestra brassicae during pupation. The DA levels were significantly higher in haemolymph, integument and brain-central nervous system of diapause-destined pupae than in non-diapause-destined pupae. The elevated level of the integumental DA content was demonstrated to be due to an increase in dopa decarboxylase activity in diapausing pupal integuments through an enhancement of transcript levels of this enzyme. Elevation of the DA level accomplished by feeding L-DOPA to last instar larvae induced a diapause-like state in more than 50% of the pupae under long daylengths.

Adaptation, Physiological↗
PubMed2001

Cold hardiness in summer and winter diapause and post-diapause pupae of the cabbage armyworm, Mamestra brassicae L. under temperature acclimation.

Cold hardiness and biochemical changes were investigated in winter and summer pupae of the cabbage armyworm Mamestra brassicae at the diapause and post-diapause stages under temperature acclimation. Diapause pupae were successively acclimated to 25, 20 and then 10 degrees C (warm-acclimated group). Pupae at the diapause and post-diapause stages were successively acclimated to 5, 0, -5 and then -10 degrees C (cold-acclimated groups). Supercooling point values in winter and summer pupae remained constant regardless of the diapause stages and acclimated temperatures. Warm-acclimated pupae at the diapause stage did not survive the subzero temperature exposure, whereas, cold-acclimated pupae achieved cold hardiness to various degrees. Winter pupae were more cold hardy than summer pupae, and pupae at the post-diapause stage were more cold hardy than those at the diapause stage. Trehalose contents in winter pupae rose under cold acclimation. Summer pupae accumulated far lower trehalose contents than winter pupae, with the maximal level occurring in winter pupae at the post-diapause stage. Glycogen content remained at a high level in diapause pupae after warm acclimation, whereas it decreased after cold acclimation. Alanine, the main free amino acid in haemolymph after cold acclimation, increased at lower temperatures in both diapause and post-diapause pupae, but the increase was greater in the diapause pupae. These results suggest that cold hardiness is more fully developed in winter pupae than in summer pupae, and cold acclimation provides higher cold hardiness in winter pupae at the post-diapause stage than at the diapause stage.

Journal Article↗
PubMed2002

The role of photoperiod and temperature in determination of summer and winter diapause in the cabbage beetle, Colaphellus bowringi (Coleoptera: Chrysomelidae).

The cabbage beetle, Colaphellus bowringi, is a short-day species undergoing an imaginal summer and winter diapause. Its photoperiodic response highly depends on temperature. All adults entered diapause at </= 20 degrees C regardless of photoperiods. High temperatures strongly weakened the diapause-inducing effects of long daylengths. The diapause-averting influence of short daylengths was expressed only at high temperatures (above 20 degrees C). This indicates that the beetle has a cryptic ability to reproduce in summer. In fact, summer and winter diapause were induced principally by relatively low temperatures in the field, whereas photoperiod had less influence on diapause induction. The critical daylength for the autumnal population was between 12 h and 13 h. By transferring from a long day to a short day or vice versa at different times after hatching, it was shown that the sensitive stage with regard to photoperiod was the larva, whereas a long day was photoperiodically more potent than a short day. The sensitive stage to temperature encompassed the larval, pupal and adult stages. This different response pattern serves to ensure that the beetle enters summer and winter diapause in time. The selections for non-diapause trait under laboratory (at 25 degrees C) and natural conditions (at >24 degrees C) showed that the beetle could lose its sensitivity to photoperiod very rapidly.

Journal Article↗
PubMed1997

Does PBAN play an alternative role of controlling pheromone emission in the cabbage looper moth, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae)?

There is an active process by which sex pheromone reserves of female cabbage looper moths, Trichoplusia ni, are transported to the gland's surface during the nocturnal period of calling. We hypothesized that this mobilization was controlled by a head factor, possibly related to the pheromone biosynthesis activating neuropeptides (PBAN) that in other species stimulate pheromone synthesis. We evaluated the impact of head extracts of T. ni on pheromone emission and glandular content of pheromone. During the photophase injected head extracts stimulated an increased pheromone emission rate in females, but glandular content of pheromone was not affected. Head extracts of H. virescens, a species with known PBAN activity, and synthetic PBAN stimulated an increased pheromone emission rate in T. ni. There was some specificity of the response of female T. ni to PBAN, in that several other unrelated polypeptides did not stimulate this type of response. Previously it had been determined that brain factors do not play a role in stimulating pheromone biosynthesis in T. ni. Our results indicate that there may be additional avenues by which PBAN or related neuropeptides control pheromone emission, including transport of pheromone reserves to the surface of the sex pheromone gland.

Journal Article↗
PubMed1997

Summer and winter diapause in pupae of the cabbage butterfly, Pieris melete Ménétriés.

The cabbage butterfly, Pieris melete is multivoltine with a pupal summer and winter diapause. Summer and winter diapause are induced principally by relatively long and short daylengths, respectively. The intermediate to relatively short daylengths of autumn permitted some pupae to develop without diapause in the field. A short daylength had a stronger diapause inducing effect than a relatively long one under higher temperatures. The principal sensitive phase for photoperiodic response occurred before the late 3rd larval instar. The critical daylength for wild autumnal populations was between 12h 30min and 12h 40min at an average temperature of 20.5 degrees C. A night interruption by 2h of light averted diapause most effectively when it was placed 10 to 12h after lights-off. High temperatures and long days during summer inhibited the incidence of diapause, suggesting that the occurrence of summer diapause is due to the specific climatic conditions occurring in April and early May, rather than to the high temperatures in summer. This indicates that the butterfly has a cryptic ability to reproduce in summer. High temperatures delayed diapause development, whereas low temperatures enhanced it, indicating that the optimum temperature of diapause development is lower. The diapause regulating mechanisms thus ensure that the species synchronises its development and reproduction with the growth seasons of the host plants and provide the species with a high degree of flexibility in its life cycle.

Journal Article↗
PubMed1998

Two strains of cabbage seed weevil (Coleoptera: Curculionidae) exhibit differential susceptibility to a transgenic oilseed rape expressing oryzacystatin I.

The aim of this study was to assess the potential effect of a transgenic line of oilseed rape expressing oryzacystatin I (OCI) on two strains of cabbage seed weevil. The level of OCI expression in seeds was approximately 0.05% of total soluble proteins. The insects were field-collected in two different locations, and their progeny was analyzed after a 3 week-development in pods. Both strains showed a similar pattern of proteolytic activity, and similar levels of OCI-sensitive proteinase activity in vitro. However, the larvae showed differential susceptibility to the transgenic plants. Despite inhibition of digestive proteinases in vitro by OCI in both strains, one strain showed an increased growth rate when fed the transgenic seeds, while the other strain remained unaffected. While suggesting the importance of studying individuals from different populations when assessing the effect of proteinase inhibitor-expressing plants on insect growth, our results also point out the necessity of studying the biochemical interactions taking place in vivo between the recombinant inhibitors and their target proteinases.

Journal Article↗
PubMed1999

Identification of the gland secreting oviposition-deterring pheromone in the cabbage seed weevil, Ceutorhynchus assimilis, and the mechanism of pheromone deposition.

After laying an egg into a pod of Brassica napus, the female cabbage seed weevil, Ceutorhynchus assimilis, brushes the caudal setae of the eighth abdominal tergite (VIII UT) on the host pod as she walks along it, depositing oviposition-deterring pheromone (ODP). The VIII UT is periodically extended and withdrawn, thus repeatedly rubbing against the posterior fold of the seventh urotergite (VII UT) which bears the individual outlets of glandular epidermal cells. In post-diapause, sexually mature, gravid (i.e. oviferous) females (virgin or mated) the cells of this VII UT gland were hypertrophic, showing intense secretory activity. Extracts of VII UT from these individuals elicited strong electrophysiological responses from antennal club gustatory sensilla and deterred oviposition. In pre-diapause (sexually immature) females, the cells of the VII UT gland were neither hypertrophic nor active and an extract of their VII UT elicited no significant electrophysiological or behavioural response. Extract of female rectum was a less potent oviposition deterrent than VII UT extract and elicited an electrophysiological response similar to male rectum extract. An extract of ovarian calyces and ovaries elicited no behavioural response. We conclude that ODP is secreted by the epidermal cells of the VII UT posterior fold.

Journal Article↗
PubMed1998

Kaempferol tetraglucosides from cabbage leaves.

Four flavonol glycosides were isolated from a leaf extract of cabbage and characterized by chemical and spectroscopic methods including 1H and 13C NMR and negative ion FAB-MS. The common structure of the four compounds was kaempferol 3-O-beta-D-[beta-D-glucopyranosyl(1-->2)glucopyranoside]-7- O-beta-D-[beta-D-glucopyranosyl(1-->4)glucopyranoside]. This compound was found unmodified or acylated at C-2"' (outer glucose in sophorosyl moiety) with either sinapic acid, ferulic acid or caffeic acid. The possible role of diversity in glycosylation and acylation patterns of flavonol glycosides for plant defences against herbivores is discussed.

Brassica↗