Cleavage of the Carboxyl-Terminus of LEACS2, a Tomato 1-Aminocyclopropane-1-Carboxylic Acid Synthase Isomer, by a 64-kDa Tomato Metalloprotease Produces a Truncated but Active Enzyme

1-Aminocyclopropane-1-carboxylic acid （ACC） synthase （ACS） is the principal enzyme in phytohormone ethylene biosynthesis. Previous studies have shown that the hypervariable C-terminus of ACS is proteolytically processed in vivo. However, the protease responsible for this has not yet been identified. In the present study, we investigated the processing of the 55-kDa full-length tomato ACS （LeACS2） into 52-, 50- and 49-kDa truncated isoforms in ripening tomato （Lycopersicon esculentum Mill. cv. Cooperation 903） fruit using the sodium dodecyl sulfate-boiling method. Meanwhile, an LeACS2-processing protease was purified via multi-step column chromatography from tomato fruit. Subsequent biochemical analysis of the 64-kDa purified protease revealed that it is a metalloprotease active at multiple cleavage sites within the hypervariable C-terminus of LeACS2. N-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight analysis indicated that the LeACS2-processing metalloprotease cleaves at the C-terminal sites Lys^438, Glu^447, Lys^448, Asn^456, Ser^460, Ser^462, Lys^463, and Leu^474, but does not cleave the N- terminus of LeACS2. Four C-terminus-deleted （26-50 amino acids） LeACS2 fusion proteins were overproduced and subjected to proteolysis by this metalloprotease to identify the multiple cleavage sites located on the N-terminal side of the phosphorylation site Ser^460. The results indisputably confirmed the presence of cleavage sites within the region between the α-helix domain （H14） and Ser^460 for this metalloprotease. Furthermore, the resulting C-terminally truncated LeACS2 isoforms were active enzymatically. Because this protease could produce LeACS2 isoforms in vitro similar to those detected in vivo, it is proposed that this metalloprotease may be involved in the proteolysis of LeACS2 in vivo.     

Binding to serine 65‐phosphorylated ubiquitin primes Parkin for optimal PINK1‐dependent phosphorylation and activation

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser⁶⁵)—which lies within its ubiquitin‐like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser⁶⁵. How Ser⁶⁵‐phosphorylated ubiquitin (ubiquitin^{Phospho‐Ser65}) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin^{Phospho‐Ser65} binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser⁶⁵ by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site‐directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin^{Phospho‐Ser65}, thereby promoting Parkin Ser⁶⁵ phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser⁶⁵ phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin^{Phospho‐Ser65} to Parkin disrupts the interaction between the Ubl domain and C‐terminal region, thereby increasing the accessibility of Parkin Ser⁶⁵. Finally, purified Parkin maximally phosphorylated at Ser⁶⁵ in vitro cannot be further activated by the addition of ubiquitin^{Phospho‐Ser65}. Our results thus suggest that a major role of ubiquitin^{Phospho‐Ser65} is to promote PINK1‐mediated phosphorylation of Parkin at Ser⁶⁵, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho‐Ser⁶⁵‐binding pocket on the surface of Parkin that is critical for the ubiquitin^{Phospho‐Ser65} interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin^{Phospho‐Ser65}, which could aid in the development of Parkin activators that mimic the effect of ubiquitin^{Phospho‐Ser65}.     

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure

Cry2Ab, a pore‐forming toxin derived from Bacillus thuringiensis, is widely used as a bio‐insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250‐kDa prepore oligomer. Site‐directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.     

Identification of phosphorylation sites in the COOH‐terminal tail of the μ‐opioid receptor

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C‐terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK‐293) cells. Under basal conditions, MOPr is phosphorylated on Ser³⁶³ and Thr³⁷⁰, while in the presence of morphine or [D‐Ala2, NMe‐Phe4, Gly‐ol5]‐enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser³⁵⁶, Thr³⁵⁷ and Ser³⁷⁵. Using N‐terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C‐terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein‐coupled receptor kinase 2 (GRK2) phosphorylates Ser³⁷⁵, protein kinase C (PKC) phosphorylates Ser³⁶³, while CaMKII phosphorylates Thr³⁷⁰. Phosphorylation of the GST fusion protein of the C‐terminal tail of MOPr enhanced its ability to bind arrestin‐2 and ‐3. Hence, our study identifies both the basal and agonist‐stimulated phospho‐acceptor sites in the C‐terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.     

Multiple signaling pathways in bovine chromaffin cells regulate tyrosine hydroxylase phosphorylation at Ser19, Ser31, and Ser40

Intact bovine adrenal medullary chromaffin cells were preincubated with³²PO₄, and the multiplesite phosphorylation of tyrosine hydroxylase (TH) was studied. Up to eight³²P-labeled peptides were produced by tryptic hydrolysis of TH; however, all of the tryptic phosphopeptides were derived from four phosphorylation sites—Ser⁸, Ser¹⁹, Ser³¹ and Ser⁴⁰. In situ regulation of³²P incorporation into the latter three sites was demonstrated with a diverse set of pharmacological agents.³²P incorporation into Ser¹⁹ was preferentially increased by brief exposures to depolarizing secretagogues. Longer treatments also increased Ser³¹ and Ser⁴⁰ phosphorylation. Nicotine, muscarine and vasoactive intestinal polypeptide—reflecting cholinergic and non-cholinergic components of sympatho-adrenal transmission—each produced different patterns of multiple-site phosphorylation of TH. Nicotine, bradykinin and histamine increased³²P incorporation at each of the three sites whereas muscarine, angiotensin II, endothelin III, prostaglandin E1, GABA and ATP selectively increased Ser³¹ phosphorylation. Nerve growth factor did not influence TH phosphorylation in chromaffin cells from adult adrenal glands but selectively increased Ser³¹ phosphorylation in chromaffin cells isolated from calf adrenal glands.³²P incorporation into Ser⁴⁰ was selectively increased by forskolin and other cAMP-acting agents whereas vasoactive intestinal polypeptide increased Ser³¹ and Ser⁴⁰ phosphorylation. Thus, the phosphorylation of TH in bovine chromaffin cells appears to be regulated at three sites by three separate intracellular signaling pathways—Ser¹⁹ via Ca²⁺/calmodulin-dependent protein kinase II; Ser³¹ via ERK (MAP2 kinases); and Ser⁴⁰ via cAMP-dependent protein kinase. These signaling pathways, as well as the extracellular signals that were effective in stimulating them, are similar to those previously described for TH in rat pheochromocytoma cells. However, several of the pharmacological agents produced different patterns of multiple-site TH phosphorylation in the bovine chromaffin cells. These differences between tissues could be accounted for by differences in the coupling/access between the extracellular signal transduction systems and the intracellular signaling pathways as opposed to differences in the intracellular signaling pathwaysper se.Special issue dedicated to Dr. Paul Greengard     

<Emphasis Type="Italic">Cotesia plutellae</Emphasis> bracovirus suppresses expression of an antimicrobial peptide,cecropin, in the diamondback Moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis>, challenged by bacteria

An endoparasitoid wasp, Cotesia plutellae, induces significant immunosuppression of host insect, Plutella xylostella. This study was focused on suppression in humoral immune response of P. xylostella parasitized by C. plutellae. An EST database of P. xylostella provided a putative cecropin gene (PxCec) which is 627 bp long and encodes 66 amino acids. A signal peptide (22 amino acids) is predicted and two putative O-glycosylation sites in threonine are located at positions 58 and 64. Without bacterial infection, PxCec was expressed in pupa and adult stages but not in the egg and larval stages. Upon bacterial challenge, however, the larvae expressed PxCec as early as 3 h post infection (PI) and maintained high expression levels at 12–24 h PI. By 48 h PI, its expression noticeably diminished. All tested tissues of bacteria-infected P. xylostella showed PxCec expression. However, other microbes, such as virus and fungus, did not induce the PxCec expression. Parasitization by C. plutellae suppressed the expression of PxCec in response to bacterial challenge. Among the parasitic factors of C. plutellae, its symbiotic virus (C. plutellae bracovirus: CpBV) alone was able to inhibit the expression of PxCec of P. xylostella challenged by bacteria. These results indicate that PxCec expression is regulated by both immune and developmental processes in P. xylostella. The parasitization by C. plutellae inhibited the expression of PxCec by the wasp’s symbiotic virus.     

Dephosphorylation of clustered phosphoserine residues in human Grb14 by protein phosphatase 1 and its effect on insulin receptor complex formation

The physical interaction of the human growth factor receptor‐bound protein 14 (hGrb14) and the insulin receptor (IR) represses insulin signaling. With respect to the recruiting mechanism of hGrb14 to IR respond to insulin stimulus, our previous reports have suggested that phosphorylation of Ser³⁵⁸, Ser³⁶², and Ser³⁶⁶ in hGrb14 by glycogen synthase kinase‐3 repressed hGrb14–IR complex formation. In this study, we investigated phosphatase‐mediated dephosphorylation of the hGrb14 phosphoserine residues. An in vitro phosphatase assay with hGrb14‐derived synthetic phosphopeptides suggested that protein phosphatase 1 (PP1) is involved in the dephosphorylation of Ser³⁵⁸ and Ser³⁶². Furthermore, coimmunoprecipitation experiments suggested that insulin‐induced hGrb14–IR complex formation was repressed by the substitution of Ser³⁵⁸ or Ser³⁶² with glutamic acid. These findings suggested that phosphate groups on Ser³⁵⁸ and Ser³⁶² in hGrb14 are dephosphorylated by PP1, and the dephosphorylation facilitates hGrb14–IR complex formation.     

Baculoviral p94 homologs encoded in Cotesia plutellae bracovirus suppress both immunity and development of the diamondback moth,Plutellae xylostella

Polydnaviruses (PDVs) are a group of insect DNA viruses, which exhibit a mutual symbiotic relationship with their specific host wasps. Moreover, most encapsidated genes identified so far in PDVs share homologies with insect‐originated genes, but not with virus‐originated genes. In the meantime, PDVs associated with 2 wasp genera Cotesia and Glytapanteles encode some genes presumably originated from other viruses. Cotesia plutellae bracovirus (CpBV) encodes 4 genes homologous to baculoviral p94: CpBV‐E94k1, CpBV‐E94k2, CpBV‐E94k3, and CpBV‐E94k4. This study was conducted to predict the origin of CpBV‐E94ks by comparing their sequences with those of baculoviral orthologs and to determine the physiological functions by their transient expressions in nonparasitized larvae and subsequent specific RNA interference. Our phylogenetic analysis indicated that CpBV‐E94ks were clustered with other E94ks originated from different PDVs and shared high similarity with betabaculoviral p94s. These 4 CpBV genes were expressed during most developmental stages of the larvae of Plutella xylostella parasitized by C. plutellae. Expression of these 4 E94ks was mainly detected in hemocytes and fat body. Subsequent functional analysis by in vivo transient expression showed that all 4 viral genes significantly inhibited both host immune and developmental processes. These results suggest that CpBV‐E94ks share an origin with betabaculoviral p94s and play parasitic roles in suppressing host immune and developmental processes.     

Isolation and characterization of Pseudomonas cedrina infecting Plutella xylostella (Lepidoptera: Plutellidae)

The diamondback moth, Plutella xylostella, is one of the most destructive pests worldwide and its management relies exclusively on frequent application of chemical insecticides. Resistance to common insecticides is now widespread, and novel classes of insecticides are needed. Entomopathogenic bacteria and their related products play an important role in the management of this pest. In the present work, one bacterial strain was separated from infected pupae of P. xylostella collected from field and its pathogenicity was evaluated. On the basis of the 16S ribosomal RNA sequencing, BLASTN, and phylogenetic analysis, this bacterial isolate was identified as Pseudomonas cedrina. Oral administration of P. cedrina at levels above 10,000 CFU/ml gave significant mortality to P. xylostella larvae. The pathogenicity was also observed by reduced longevity and fecundity in adult females. However, when live bacterial cells were removed, the cultured broth lost any pathogenicity. In response to the bacterial infection, P. xylostella expressed antimicrobial and stress‐associated genes. A mixture treatment of P. cedrina and Bacillus thuringiensis showed an additive effect on larval mortality of P. xylostella. These results indicated that P. cedrina is an opportunistic entomopathogen without secretion of toxins. Furthermore, the additive effect of P. cedrina and B. thuringiensis provide a new insight to develop new strategy for controlling P. xylostella.     

Expression profiles of two thaumatin‐like protein (TmTLP) genes in responses to various micro‐organisms from Tenebrio molitor

It is well known that anti‐fungal thaumatin‐like proteins (TLPs) play important roles in plants. Here, we investigated the expression analysis of thaumatin‐like protein genes TmTLPs in response to various pathogens in Tenebrio molitor. Developmental expression patterns of TmTLPs show that TmTLPs are highly expressed in the early pupal and adult stages. Furthermore, tissue‐specific expression patterns of TmTLPs indicate that TmTLP1 is highly expressed in the integument and gut of last instar larvae and the integument and Malpighian tubules of 5‐day old adults. In contrast, TmTLP2 is highly expressed in the gut of both last instar larvae and 5‐day old adults. We hypothesize that the expression of TmTLP genes in developmental stages may be related to molting and body remodeling stresses. In addition, the induction patterns of TmTLP genes indicate that TmTLPs were slightly induced by Escherichia coli, Staphylococcus aureus, Candida albicans and Listeria monocytogenes. Furthermore, TmTLP1 and TmTLP2 were strongly induced in response to E. coli at 9 h post‐injection and L. monocytogenes at 3 h post‐injection. Our results suggest that TmTLPs may possess antimicrobial functions in T. molitor.     

Susceptibility of major lepidopterans to δ-endotoxins and a formulation of Bacillus thuringiensis (B.t.) on vegetable brassicas in Taiwan

Baseline susceptibility of Plutella xylostella, Crocidolomia binotalis and Hellula undalis to Bacillus thuringiensis (B.t.) δ-endotoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca) and a formulation (Xentari®) was assessed using a leaf-dip bioassay method. The toxins Cry1Ac, Cry1Aa and Cry1Ca were equally toxic to P. xylostella. Crocidolomia binotalis was highly susceptible to all Cry1A toxins and it was least sensitive to Cry1Ca. Conversely, H. undalis was highly sensitive to Cry1Ca, but less susceptible to Cry1A toxins. Hence, the susceptibility of H. undalis to Xentari®, a formulation containing Cry1C as a major toxin, was significantly higher than C. binotalis or P. xylostella.     

Bacteria abundance and diversity of different life stages of Plutella xylostella (Lepidoptera: Plutellidae), revealed by bacteria culture‐dependent and PCR‐DGGE methods

Microbial abundance and diversity of different life stages (fourth instar larvae, pupae and adults) of the diamondback moth, Plutella xylostella L., collected from field and reared in laboratory, were investigated using bacteria culture‐dependent method and PCR‐DGGE analysis based on the sequence of bacteria 16S rRNA V3 region gene. A large quantity of bacteria was found in all life stages of P. xylostella. Field population had higher quantity of bacteria than laboratory population, and larval gut had higher quantity than pupae and adults. Culturable bacteria differed in different life stages of P. xylostella. Twenty‐five different bacterial strains were identified in total, among them 20 strains were presented in larval gut, only 8 strains in pupae and 14 strains in adults were detected. Firmicutes bacteria, Bacillus sp., were the most dominant species in every life stage. 15 distinct bands were obtained from DGGE electrophoresis gel. The sequences blasted in GenBank database showed these bacteria belonged to six different genera. Phylogenetic analysis showed the sequences of the bacteria belonged to the Actinobacteri, Proteobacteria and Firmicutes. Serratia sp. in Proteobacteria was the most abundant species in larval gut. In pupae, unculturable bacteria were the most dominant species, and unculturable bacteria and Serratia sp. were the most dominant species in adults. Our study suggested that a combination of molecular and traditional culturing methods can be effectively used to analyze and to determine the diversity of gut microflora. These known bacteria may play important roles in development of P. xylostella.     

Pharmacological characterization of a β‐adrenergic‐like octopamine receptor in Plutella xylostella

The β‐adrenergic‐like octopamine receptor (OA2B2) belongs to the class of G‐protein coupled receptors. It regulates important physiological functions in insects, thus is potentially a good target for insecticides. In this study, the putative open reading frame sequence of the Pxoa2b2 gene in Plutella xylostella was cloned. Orthologous sequence alignment, phylogenetic tree analysis, and protein sequence analysis all showed that the cloned receptor belongs to the OA2B2 protein family. PxOA2B2 was transiently expressed in HEK‐293 cells. It was found that PxOA2B2 could be activated by both octopamine and tyramine, resulting in increased intracellular cyclic AMP (cAMP) levels, whereas dopamine and serotonin were not effective in eliciting cAMP production. Further studies with series of PxOA2B2 agonists and antagonists showed that all four tested agonists (e.g., naphazoline, clonidine, 2‐phenylethylamine, and amitraz) could activate the PxOA2B2 receptor, and two of tested antagonists (e.g., phentolamine and mianserin) had significant antagonistic effects. However, antagonist of yohimbine had no effects. Quantitative real‐time polymerase chain reaction analysis showed that Pxoa2b2 gene was expressed in all developmental stages of P. xylostella and that the highest expression occurred in male adults. Further analysis with fourth‐instar P. xylostella larvae showed that the Pxoa2b2 gene was mainly expressed in Malpighian tubule, epidermal, and head tissues. This study provides both a pharmacological characterization and the gene expression patterns of the OA2B2 in P. xylostella, facilitating further research for insecticides using PxOA2B2 as a target.     

Interdependent Phosphorylation within the Kinase Domain T-loop Regulates CHK2 Activity

Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser¹⁹, Ser³³, Ser³⁵, and Thr⁶⁸. This precedes autophosphorylation of Thr³⁸³/Thr³⁸⁷ in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366–406). We report four novel phosphorylation sites at Ser³⁷², Thr³⁷⁸, Thr³⁸⁹, and Tyr³⁹⁰. Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr³⁸⁹ was a prerequisite to phosphorylation at Thr³⁸⁷ but not at Thr³⁸³. Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser³⁷⁹, Thr³⁸⁹, and Thr³⁸³/Thr³⁸⁹. Phosphorylation at Tyr³⁹⁰ was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr³⁷², Thr³⁷⁹, Thr³⁸³, Thr³⁸⁹, Thr³⁸³/Thr³⁸⁷, and Thr³⁸³/Thr³⁸⁹. Chk2 hyper-phosphorylated species at Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹ and Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹/Tyr³⁹⁰ relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2.     

Expression of myostatin in the spotted rose snapper Lutjanus guttatus during larval and juvenile development under cultured conditions

In this study, the developmental expression pattern of myostatin (mstn) in the spotted rose snapper Lutjanus guttatus under culture conditions is presented. The full coding sequence of mstn from L. guttatus was isolated from muscle tissue, obtaining 1134 nucleotides which encode a peptide of 377 amino acids. The phylogenetic analysis indicated that this sequence corresponds to mstn‐1. mstn expression was detected in embryonic stages, and maintained at low levels until 28 days post‐hatch, when it showed a significant increase, coinciding with the onset of metamorphosis. After that, expression was fluctuating, coinciding probably with periods of rapid and slow muscle growth or individual growth rates. mstn expression was also analysed by body mass with higher levels detected in smaller animals, irrespective of age. mstn was also expressed in other tissues from L. guttatus, presenting higher levels in brain, eye and gill. In brain for instance, two variants of mstn were isolated, both coding sequences were identical to muscle, except that one of them contained a 75 nucleotide deletion in exon 1, maintaining the reading frame but deleting two conserved cysteine residues. Phylogenetic analysis indicated that this brain variant was also mstn‐1. The function of this variant is not clear and needs further investigation. These results indicate that mstn‐1 participates in different physiological processes other than muscle growth in fishes.     

Downregulation of ubiquitin gene expression in the deltamethrin‐resistant diamondback moth (Plutella xylostella L.)

Plutella xylostella (L.) has long been regarded as a very important universal pest of cruciferous crops. It is therefore important to understand its mechanisms of insecticide resistance. In our previous study, we obtained a differently expressed sequence with relatively high homology to the ubiquitin (UBI) gene as assessed via cDNA representational difference analysis (RDA). To further study this product, we cloned UBI open reading frame (ORF) sequences identified from both sexes of the deltamethrin‐resistant strain (DRF and DRM) and the deltamethrin‐sensitive strain (SF and SM) of P. xylostella. The deduced protein sequence of DRM was different from the other samples. The expression level was measured using real‐time quantitative polymerase chain reaction (PCR) and Western blot. The results of both analyses revealed the expression levels, in order from highest to lowest, to be in SF, SM, DRF and DRM. This indicates that the long‐term use of deltamethrin on P. xylostella results in decreased expression of the UBI gene.     

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation

The neuron-specific cation chloride cotransporter KCC2 plays a crucial role in hyperpolarizing synaptic inhibition. Transporter dysfunction is associated with various neurological disorders, raising interest in regulatory mechanisms. Phosphorylation has been identified as a key regulatory process. Here, we retrieved experimentally observed phosphorylation sites of KCC2 from public databases and report on the systematic analysis of six phosphorylated serines, Ser²⁵, Ser²⁶, Ser⁹³⁷, Ser¹⁰²², Ser¹⁰²⁵, and Ser¹⁰²⁶. Alanine or aspartate substitutions of these residues were analyzed in HEK-293 cells. All mutants were expressed in a pattern similar to wild-type KCC2 (KCC2^WT). Tl⁺ flux measurements demonstrated unchanged transport activity for Ser²⁵, Ser²⁶, Ser¹⁰²², Ser¹⁰²⁵, and Ser¹⁰²⁶ mutants. In contrast, KCC2^S937D, mimicking phosphorylation, resulted in a significant up-regulation of transport activity. Aspartate substitution of Thr⁹³⁴, a neighboring putative phosphorylation site, resulted in a comparable increase in KCC2 transport activity. Both KCC2^T934D and KCC2^S937D mutants were inhibited by the kinase inhibitor staurosporine and by N-ethylmaleimide, whereas KCC2^WT, KCC2^T934A, and KCC2^S937A were activated. The inverse staurosporine effect on aspartate versus alanine substitutions reveals a cross-talk between different phosphorylation sites of KCC2. Immunoblot and cell surface labeling experiments detected no alterations in total abundance or surface expression of KCC2^T934D and KCC2^S937D compared with KCC2^WT. These data reveal kinetic regulation of transport activity by these residues. In summary, our data identify a novel key regulatory phosphorylation site of KCC2 and a functional interaction between different conformation-changing post-translational modifications. The action of pharmacological agents aimed to modulate KCC2 activity for therapeutic benefit might therefore be highly context-specific.     

Effects of three different cultivars of cruciferous plants on the age‐stage,two‐sex life table traits of Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Plutella xylostella is an important pest of cruciferous crops worldwide. However, information regarding the age‐stage, two‐sex life parameters of P. xylostella, which is vital for designing more effective control methods, is currently lacking. The present study reports age‐stage, two‐sex life table parameters for P. xylostella on napa cabbage (Brassica oleracea var. napa), white cabbage (B. oleracea var. capitata), and cauliflower (B. oleracea var. botrytis) under laboratory conditions at 25 ± 2°C, 50–60% relative humidity, and a 16‐h light : 8‐h dark photoperiod. The time for development from an egg to a male or female adult P. xylostella on white cabbage (mean [± SE] 41.15 ± 0.54 and 39.50 ± 0.54 days, respectively) was significantly longer than that on cauliflower and napa cabbage. Furthermore, P. xylostella fecundity on cauliflower (261.90 ± 4.53 eggs female) was significantly highest than on napa cabbage and white cabbage. Intrinsic rate of increase (r) and finite rate of increase (λ) were highest on cauliflower 0.182 day^?1 and 1.199 day^?1 respectively as comparison to napa cabbage and white cabbage. The highest gross reproductive rate (GRR) and net reproductive rates (R₀) of P. xylostella 65.87 and 52.58 respectively on cauliflower then those of other hosts. The findings of the present study indicate that cauliflower is the most suitable cultivar (host) for the development of P. xylostella. Based on these findings, crops like cauliflower can be used as trap crops when napa cabbage and white cabbage are the main crops.     

The insecticidal crystal protein Cry2Ab10 from Bacillus thuringiensis: cloning, expression, and structure simulation

The cry2Ab-type gene was cloned from Bacillus thuringiensis and designated as cry2Ab10. The recombinant Cry2Ab10 protein expressed in E. coli cells shows high toxicity against Plutella xylostella. The protein structure was constructed by homology modeling, and the receptor-binding sites were predicted by a molecular docking method.