Post-ingestive stability of a mulberry Kunitz-type protease inhibitor MnKTI-1 in the digestive lumen of silkworm: dual inhibition towards α-amylase and serine protease.

BACKGROUND: Adaptation of specialist insects to their host plants and defense responses of plants to phytophagous insects have been extensively recognized while the dynamic interaction between these two events has been largely underestimated. Here, we provide evidence for characterization of an unrevealed dynamic interaction mode of digestive enzymes of specialist insect silkworm and inhibitor of its host plant mulberry tree. RESULTS: MnKTI-1, a mulberry Kunitz-type protease inhibitor, whose messenger RNA (mRNA) transcription and protein expression in mulberry leaf were severely triggered and up-regulated by tens of times in a matter of hours in response to silkworm, Bombyx mori, and other mulberry pest insects, suggesting a quick response and broad spectrum to insect herbivory. MnKTI-1 proteins were detected in gut content and frass of specialist B. mori, and exhibited significant post-ingestive stability. Recombinant refolded MnKTI-1 (rMnKTI-1) displayed binding affinity to digestive enzymes and a dual inhibitory activity to α-amylase BmAmy and serine protease BmSP2956 in digestive juice of silkworm. Moreover, data from in vitro assays proved that the inhibition of recombinant rMnKTI-1 to BmAmy can be reverted by pre-incubation with BmSP15920, an inactivated silkworm digestive protease that lack of complete catalytic triad. CONCLUSION: These findings demonstrate that mulberry MnKTI-1 has the potential to inhibit the digestive enzyme activities of its specialist insect herbivore silkworm, whereas this insect may employ inactivated proteases to block protease inhibitors to accomplish food digestion. The current work provides an insight to better understand the interacting mode between host plant Kunitz protease inhibitors and herbivorous insect digestive enzymes. © 2024 Society of Chemical Industry.

Serine protease SP7 cleaves prophenoloxidase and is regulated by two serpins in Ostrinia furnacalis melanization.

Melanization is an innate immune response in insects to defend against the invading pathogens and parasites. During melanization, prophenoloxidase (PPO) requires proteolytic activation by its upstream prophenoloxidase-activating protease (PAP). We here cloned a full-length cDNA for a serine protease, named as SP7, from Ostrinia furnacalis. The open reading frame of SP7 encodes 421-amino acid residue protein with a 19-residue signal peptide. qRT-PCR analysis showed that SP7 mRNA levels were significantly upregulated upon exposure to microbial infection. Recombinant SP7 zymogen was activated by serine protease SP2. The active SP7 could cleave O. furnacalis PPOs including PPO2, PPO1b and PPO3. Additionally, active SP7 could form covalent complexes with serine protease inhibitor serpin-3 and serpin-4. The activity of SP7 in cleaving a colorimetric substrate IEARpNA or O. furnacalis PPOs was efficiently blocked by either serpin-3 or serpin-4. Our work thus revealed that SP7 and SP2 partially constituted a PPO activation cascade in which SP7 was activated by SP2 and then likely worked as a PAP. SP7 was effectively regulated by serpin-3 and serpin-4. The results would allow further advances in the understanding of melanization mechanisms in O. furnacalis.

A beta-glucosidase of an insect herbivore determines both toxicity and deterrence of a dandelion defense metabolite.

Gut enzymes can metabolize plant defense compounds and thereby affect the growth and fitness of insect herbivores. Whether these enzymes also influence feeding preference is largely unknown. We studied the metabolization of taraxinic acid ß-D-glucopyranosyl ester (TA-G), a sesquiterpene lactone of the common dandelion (Taraxacum officinale) that deters its major root herbivore, the common cockchafer larva (Melolontha melolontha). We have demonstrated that TA-G is rapidly deglucosylated and conjugated to glutathione in the insect gut. A broad-spectrum M. melolontha ß-glucosidase, Mm_bGlc17, is sufficient and necessary for TA-G deglucosylation. Using cross-species RNA interference, we have shown that Mm_bGlc17 reduces TA-G toxicity. Furthermore, Mm_bGlc17 is required for the preference of M. melolontha larvae for TA-G-deficient plants. Thus, herbivore metabolism modulates both the toxicity and deterrence of a plant defense compound. Our work illustrates the multifaceted roles of insect digestive enzymes as mediators of plant-herbivore interactions.

Plants produce certain substances to fend off attackers like plant-feeding insects. To stop these compounds from damaging their own cells, plants often attach sugar molecules to them. When an insect tries to eat the plant, the plant removes the stabilizing sugar, 'activating' the compounds and making them toxic or foul-tasting. Curiously, some insects remove the sugar themselves, but it is unclear what consequences this has, especially for insect behavior. Dandelions, Taraxacum officinale, make high concentrations of a sugar-containing defense compound in their roots called taraxinic acid ß-D-glucopyranosyl ester, or TA-G for short. TA-G deters the larvae of the Maybug ­ a pest also known as the common cockchafer or the doodlebug ­ from eating dandelion roots. When Maybug larvae do eat TA-G, it is found in their systems without its sugar. However, it is unclear whether it is the plant or the larva that removes the sugar. A second open question is how the sugar removal process affects the behavior of the Maybug larvae. Using chemical analysis and genetic manipulation, Huber et al. investigated what happens when Maybug larvae eat TA-G. This revealed that the acidity levels in the larvae's digestive system deactivate the proteins from the dandelion that would normally remove the sugar from TA-G. However, rather than leaving the compound intact, larvae remove the sugar from TA-G themselves. They do this using a digestive enzyme, known as a beta-glucosidase, that cuts through sugar. Removing the sugar from TA-G made the compound less toxic, allowing the larvae to grow bigger, but it also increased TA-G's deterrent effects, making the larvae less likely to eat the roots. Any organism that eats plants, including humans, must deal with chemicals like TA-G in their food. Once inside the body, enzymes can change these chemicals, altering their effects. This happens with many medicines, too. In the future, it might be possible to design compounds that activate only in certain species, or under certain conditions. Further studies in different systems may aid the development of new methods of pest control, or new drug treatments.

Drosophila Rab39 Attenuates Lysosomal Degradation.

Lysosomal degradation, the common destination of autophagy and endocytosis, is one of the most important elements of eukaryotic metabolism. The small GTPases Rab39A and B are potential new effectors of this pathway, as their malfunction is implicated in severe human diseases like cancer and neurodegeneration. In this study, the lysosomal regulatory role of the single Drosophila Rab39 ortholog was characterized, providing valuable insight into the potential cell biological mechanisms mediated by these proteins. Using a de novo CRISPR-generated rab39 mutant, we found no failure in the early steps of endocytosis and autophagy. On the contrary, we found that Rab39 mutant nephrocytes internalize and degrade endocytic cargo at a higher rate compared to control cells. In addition, Rab39 mutant fat body cells contain small yet functional autolysosomes without lysosomal fusion defect. Our data identify Drosophila Rab39 as a negative regulator of lysosomal clearance during both endocytosis and autophagy.

PAP1 activates the prophenoloxidase system against bacterial infection in Musca domestica.

We previously identified three putative prophenoloxidase-activating proteinase (mdPAP1, mdPAP2, and mdPAP3) genes from housefly Musca domestica by transcriptomic analysis. In this study, mdPAP1 cDNA was cloned, and the function of its encoded protein was analyzed. The cDNA of mdPAP1 was 1358 bp, and it contained a single open reading frame of 1122 bp encoding a predicted MdPAP1 protein of 373 amino acids. The estimated molecular weight of MdPAP1 was 41267.08 Da with an isoelectric point of 6.25. The deduced amino acid sequence of MdPAP1 exhibited high similarity to known PAPs of insects. mdPAP1 was detected in larvae, pupae, and adult housefly, and the expression level of mdPAP1 was upregulated in bacterial challenged larvae. The recombinant protein of MdPAP1 expressed in Escherichia coli could cleave the prophenoloxidase into phenoloxidase in M. domestica hemolymph infected by bacteria and result in a significant increase of the total phenoloxidase activity. In addition, RNA interference-mediated gene silencing of mdPAP1 significantly increased the mortality of M. domestica larvae. Results indicated that mdPAP1 was involved in the activation of the prophenoloxidase against bacterial infection in M. domestica.

The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo.

The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLow) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.

Efficacy of maggot debridement therapy on refractory leg ulcers of Behçet disease: an open-label study.

BACKGROUND: Cutaneous ulcers of Behçet disease (BD) are rare but have high morbidity and resistance to conventional therapies. An important and essential aspect of ulcer management is debridement. Regarding maggot therapy (MT), excretions of the green bottle fly, Lucilia sericata, have been shown to have the ability to remove necrotic debris and promote healing. AIM: To evaluate the efficacy of MT for cutaneous ulcers of BD. METHODS: In this open-label trial, patients with BD with refractory leg ulcers suitable for MT were enrolled. Maggot application was performed until complete debridement was achieved, and all patients were followed up for 12 months afterwards to assess the total healing of ulcers. RESULTS: In total, 24 patients with 32 ulcers were enrolled. Using MT, 91.6% of all ulcers were completely debrided. Mean time to debridement was 14.9 days and mean number of cycles required was 5.3. Mean ulcer size was decreased by 23% with treatment. Time to debridement was positively correlated with pretreatment ulcer size and ulcer duration (P = 0.01 and P < 0.01) but not with ulcer depth, comorbidities, smoking, age or sex (P > 0.05 for all). During follow-up, 79.1% of all ulcers healed completely. Mean time required for total healing was positively correlated with ulcer duration, pretreatment and post-treatment ulcer area, ulcer depth and mean time to total debridement (P < 0.03, P = 0.00, P = 0.04 and P < 0.01, respectively). CONCLUSIONS: To our knowledge, the findings presented in this first and unique study may provide key answers about factors affecting success rate of MT in BD cutaneous ulcers.

A polybasic domain in aPKC mediates Par6-dependent control of membrane targeting and kinase activity.

Mechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells, the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity, and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.

Role of Chitin Deacetylase 1 in the Molting and Metamorphosis of the Cigarette Beetle Lasioderma serricorne.

Chitin deacetylases (CDAs) are chitin-modifying enzymes known to play vital roles in insect metamorphosis and development. In this study, we identified and characterized a chitin deacetylase 1 gene (LsCDA1) from the cigarette beetle Lasioderma serricorne. LsCDA1 contains a 1614 bp open reading frame encoding a protein of 537 amino acids that includes domain structures typical of CDAs. LsCDA1 was mainly expressed in the late larval and late pupal stages. In larval tissues, the highest level of LsCDA1 was detected in the integument. The expression of LsCDA1 was induced by 20-hydroxyecdysone (20E) in vivo, and it was significantly suppressed by knocking down the expression of ecdysteroidogenesis genes and 20E signaling genes. RNA interference (RNAi)-aided silencing of LsCDA1 in fifth-instar larvae prevented the larval-pupal molt and caused 75% larval mortality. In the late pupal stage, depletion of LsCDA1 resulted in the inhibition of pupal growth and wing abnormalities, and the expression levels of four wing development-related genes (LsDY, LsWG, LsVG, and LsAP) were dramatically decreased. Meanwhile, the chitin contents of LsCDA1 RNAi beetles were significantly reduced, and expressions of three chitin synthesis pathway genes (LsTRE1, LsUAP1, and LsCHS1) were greatly decreased. The results suggest that LsCDA1 is indispensable for larval-pupal and pupal-adult molts, and that it is a potential target for the RNAi-based control of L. serricorne.

Cadmium-induced developmental alteration and upregulation of serine-type endopeptidase transcripts in wild freshwater populations of Chironomus plumosus.

Cadmium, a toxic heavy metal, is a persistent environmental contaminant with irreversible toxicity to aquatic organisms. Chironomus plumosus, a natural species, is the largest sediment-burrowing aquatic midge in freshwater environments. In this study, we evaluated developmental defects in C. plumosus resulting from Cd exposure. In C. plumosus larvae, Cd exposure induced decreased survival and growth rates, reduction of emergence rate and sex ratio, and delayed emergence, as well as elevating the incidence of split tooth deformities. To identify potential biomarker genes to assess environmental pollutants such as Cd, we identified differentially expressed genes (DEGs) in C. plumosus exposed to various Cd concentrations. Among fourteen characterized DEGs, serine-type endopeptidase (SP) and heat shock protein 70 (HSP70) genes exhibited significant upregulation in C. plumosus larvae after Cd exposure. Therefore, we evaluated SP and HSP70 responses in natural C. plumosus populations collected from three sites of a Korean river and analyzed their correlations with eighteen environmental quality characteristics using principal component analysis. The highest expression of SP and HSP70 transcripts was observed in C. plumosus populations from Yeosu in Korea, which has high concentrations of polluting heavy metals. SP transcript expression was positively correlated with concentrations of Cd, Pb, Al, Fe, NO2, and NO3. These results suggested that environmental pollutants such as Cd can impair proteolytic activity in the digestive system of C. plumosus and may ultimately induce developmental alterations. We therefore suggest SP as a potential biomarker to assess the effects of environmental pollutants in aquatic ecosystems.

Effect of Tannic Acid on Nutrition and Activities of Detoxification Enzymes and Acetylcholinesterase of the Fall Webworm (Lepidoptera: Arctiidae).

Plant tannins, polyphenolic plant secondary metabolites are involved in important chemical defense processes in plants. In this study, tannic acid was used as the standard of plant tannins to determine the effects on nutritional indices and activities of glutathione S-transferases (GSTs), cytochrome P450 monooxygenase (CYP450), carboxylesterase (CarE), and acetylcholinesterase (AChE) in fourth-instar larvae of Hyphantria cunea (Drury) by feeding on an artificial diet containing tannic acid under different treatments. We found that tannic acid significantly affected the digestive capacity and food utilization rate of H. cunea larvae. A tannic acid concentration of less than 2.0% promoted feeding and the utilization of undesirable food by H. cunea larvae, while inhibitory effects were observed at high concentrations (>2.5%). Tannic acid had a significant effect on the activity of detoxification enzymes and AChE in H. cunea larvae in concentration-dependent and time-dependent manners (P < 0.05). These results provide new insights into the potential mechanisms underlying detoxification in H. cunea larvae against tannic acid in host plants.

Intestinal proteolytic profile changes during larval development of Anticarsia gemmatalis caterpillars.

Soybean is one of most consumed and produced grains in the world, and Anticarsia gemmatalis is a pest that causes great damage to this crop due to severe defoliation during its larval phase. Plants have mechanisms that lead to the inhibition of proteases in the intestine of these herbivores, hampering their development. Understanding this complex protease inhibitor is important for pest control. The objective of this study was to evaluate the enzymatic profiles of the intestinal proteases of the soybean caterpillar at different instars. For this, the proteolytic profile of the gut in the third, fourth, and fifth instars were analyzed. Irreversible inhibitors of proteases were separately incubated with A. gemmatalis enzyme extracts at the third, fourth, and fifth instar to assess the contribution of these proteases to total proteolytic activity. The enzymatic extracts were also evaluated with specific substrates to confirm changes in the specific activities of trypsin-like, chymotrypsin-like, and cysteine proteases at different instars. The results showed that the protease profile of A. gemmatalis gut changes throughout its larval development. The activity of cysteine proteases was more intense in the first instar. On the contrary, the serine proteases showed major activities in the late stages of the larval phase. Zymogram analysis and protein identification by liquid chromatography-mass spectrometry indicated serine protease as the main protease class expressed in the fifth instar. These results may shift the focus from the rational development of the protease inhibitor to A. gemmatalis and other Lepidoptera, as the expression of major proteases is not constant.

Characterization of a chymotrypsin-like enzyme from Trichinella spiralis and its facilitation of larva penetration into the host's enteral epithelial cells.

The aim of this work was to identify the molecular characteristics of a chymotrypsin-like enzyme from Trichinella spiralis (Tschy) and its facilitation of larval penetration into enteral epithelial cells (EECs). The complete Tschy cDNA sequence was cloned and expressed in Escherichia coli BL21. RT-PCR, IIFA and western blotting showed that Tschy was expressed at the T. spiralis muscle larvae (ML), intestinal infective L1 larvae (IL1), adult worms (AW) and embryo stages and was primarily located in the stichosome of this parasite. The results of ELISA, IIFA and Far-western assays showed that there was a specific binding between rTschy and EECs, and the binding was dependent on the dose of both rTschy and EEC proteins. Confocal microscopy demonstrated that the binding was located in the EEC cytoplasm. rTschy facilitated T. spiralis larval penetration of EECs, and anti-rTschy antibodies impeded the larval intrusion of EECs. These results demonstrate that Tschy facilitated the larval intrusion of the host's enteral epithelium and could be a candidate molecular target for vaccine against the enteral invasive phase of T. spiralis.

A light-up near-infrared probe with aggregation-induced emission characteristics for highly sensitive detection of alkaline phosphatase.

Developing activatable near-infrared (NIR) probes to specifically monitor and visualize the activities of cancer-related enzymes is highly significant yet challenging in early cancer diagnosis. Taking advantage of the unique photophysical characteristics of aggregation-induced emission (AIE) fluorophores, here we design and synthesize a novel activatable probe QMTP by conjugating an AIE fluorophore quinolone-malononitrile to a hydrophilic phosphate-modified phenol group. The probe was initially non-fluorescent in aqueous solution due to its good water solubility, but was readily activated to generate a strong NIR fluorescence upon treatment with alkaline phosphatase (ALP), which enables specific detection of ALP activity. Furthermore, we have employed QMTP to monitor and spatially map the activity of endogenous ALP both in cancer cells and in drug-treated zebrafish larvae. The experimental results reveal that the QMTP probe has great specificity and sensitivity for ALP detection. We thus believe that our work offers a promising tool for accurate detection of ALP-associated diseases in preclinical applications.

Evaluation of the sensitivity of Microhyla fissipes tadpoles to aqueous cadmium.

Cadmium (Cd) exposure is harmful to amphibians in natural environments and the Cd concentration is a key parameter in water monitoring. Cd pollution has been a severe issue in the Yangtze River and its southern reaches in recent years. Acute toxicity assays were employed to determine the tolerance limits of Cd for Microhyla fissipes tadpoles and five different concentrations of Cd (0, 50, 100, 200 and 300 µg/L) were involved to detect its chronic effects on metamorphosis, growth, locomotion, genotoxicity and enzymatic activities of M. fissipes tadpoles. The results showed that the 24-h and 48-h LC50 values of Cd on M. fissipes tadpoles were 2591.3 µg/L and 1567.9 µg/L, respectively, and the presumable non-lethal concentration obtained was 172.2 µg/L. During the 70-day chronic toxicity assays, Cd showed negative impacts on survival, growth, metamorphosis and the frequency of erythrocytes nuclear abnormality of M. fissipes tadpoles. However, the Cd exposure caused the increased body size and condition of tadpoles at complete metamorphosis (GS46). The tadpoles exposed to 200 µg/L of Cd exhibited degraded locomotor performance at GS46. Weight increments of tadpoles were inhibited at Day 14 and massive deaths were observed over the next 14 days. The enzymatic activities of tadpoles experienced a shock response stage (GS30-GS35) and a complete recovery stage (GS36-GS41) in all treatments. However, the enzymatic activities (except alkaline phosphatase) of tadpoles at GS46 increased after Cd exposure, especially at high concentrations. In summary, Cd is a threat to M. fissipes tadpoles as that causes reduced fitness.

Flumequine-Mediated Upregulation of p38 MAPK and JNK Results in Melanogenesis in B16F10 Cells and Zebrafish Larvae.

Flumequine is a well-known second generation quinolone antibiotic that induces phototoxicity. However, the effect of flumequine on skin melanogenesis is unclear. Therefore, we, for the first time, investigated whether flumequine regulates melanogenesis. The present study showed that flumequine slightly inhibited in vitro mushroom tyrosinase activity but significantly increased extracellular and intracellular melanin content in B16F10 cells and promoted the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Additionally, flumequine remarkably increased melanin pigmentation in zebrafish larvae without any toxicity. We also found that flumequine stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation; inhibition of p38 MAPK and JNK resulted in significant downregulation of extracellular and intracellular melanin content in B16F10 cells and pigmentation of zebrafish larvae accompanied with suppression of MITF and tyrosinase expression, indicating that flumequine-mediated p38 and JNK promote melanogenesis in vitro and in vivo. According to the molecular docking prediction, flumequine targeted dual-specificity MAPK phosphatase 16 (DUSP16), which is a major negative regulator of p38 MAPK and JNK. Our findings demonstrate that flumequine induces an increase in melanin content in B16F10 cells and zebrafish larvae by activating p38 MAPK and JNK. These data show the potential of flumequine for use as an anti-vitiligo agent.

Salvador-Warts-Hippo pathway regulates sensory organ development via caspase-dependent nonapoptotic signaling.

The fundamental roles for the Salvador-Warts-Hippo (SWH) pathway are widely characterized in growth regulation and organ size control. However, the function of SWH pathway is less known in cell fate determination. Here we uncover a novel role of the SWH signaling pathway in determination of cell fate during neural precursor (sensory organ precursor, SOP) development. Inactivation of the SWH pathway in SOP of the wing imaginal discs affects caspase-dependent bristle patterning in an apoptosis-independent process. Such nonapoptotic functions of caspases have been implicated in inflammation, proliferation, cellular remodeling, and cell fate determination. Our data indicate an effect on the Wingless (Wg)/Wnt pathway. Previously, caspases were proposed to cleave and activate a negative regulator of Wg/Wnt signaling, Shaggy (Sgg)/GSK3ß. Surprisingly, we found that a noncleavable form of Sgg encoded from the endogenous locus after CRISPR-Cas9 modification supported almost normal bristle patterning, indicating that Sgg might not be the main target of the caspase-dependent nonapoptotic process. Collectively, our results outline a new function of SWH signaling that crosstalks to caspase-dependent nonapoptotic signaling and Wg/Wnt signaling in neural precursor development, which might be implicated in neuronal pathogenesis.

Glucose-6-phosphate dehydrogenase is posttranslationally regulated in the larvae of the freeze-tolerant gall fly, Eurosta solidaginis, in response to freezing.

The freeze-tolerant larvae of the goldenrod gall fly (Eurosta solidaginis) undergo substantial alterations to their molecular physiology during the winter including the production of elevated quantities of glycerol and sorbitol, which function as cryoprotectants to survive whole body freezing. Production of these cryoprotectants depends on cytosolic pools of nicotinamide adenine dinucleotide phosphate H (NADPH), a major source being the pentose phosphate pathway (PPP). Glucose-6-phosphate dehydrogenase (G6PDH) mediates the rate-limiting and committed step of the PPP and therefore its molecular properties were explored in larvae sampled from control versus frozen states. G6PDH was purified from control (5°C) and frozen (-15°C) E. solidaginis larvae by a single-step chromatography method utilizing 2',5'-ADP agarose and analyzed to determine its enzymatic parameters. Studies revealed a decrease in Km for G6P in the frozen animals (to 50% of control values) suggesting an increased flux through the PPP. Immunoblotting of the purified enzyme showed differences in the relative extent of several posttranslational modifications, notably ubiquitination (95% decrease in frozen larvae), cysteine nitrosylation (61% decrease), threonine (4.1 fold increase), and serine phosphorylation (59% decrease). Together these data suggested that the increased flux through the PPP needed to generate NADPH for cryoprotectants synthesis is regulated, at least in part, through posttranslational alterations of G6PDH.

A Novel Adenosine Kinase from Bombyx mori: Enzymatic Activity, Structure, and Biological Function.

Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% ß-strand structures, respectively. The structure of BmADK was stable in pH 5.0-11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.

Effect of fluoranthene on antioxidative defense in different tissues of Lymantria dispar and Euproctis chrysorrhoea larvae.

This study examined the effect of long-term exposure to environmentally relevant concentrations of dietary fluoranthene (6.7 and 67 ng / g dry food weight) on defense mechanisms of the polyphagous forest insects Lymantria dispar L. and Euproctis chrysorrhoea L. The activities and expression of isoforms of superoxide dismutase (SOD) and catalase (CAT), the activities of glutathione S-transferase (GST) and glutathione reductase (GR), and total glutathione content (GSH) were determined in the whole midgut and midgut tissue, while SOD and CAT activities were assessed in hemolymph of the larvae. The results showed significant changes of enzyme activities, with more pronounced responses in larval midgut tissues, and between-species differences in patterns of response. Significantly increased activity of SOD was recorded in the whole midgut and midgut tissue of L. dispar larvae, as well as in midgut tissue of E. chrysorrhoea larvae. Fluoranthene increased CAT activity in midgut tissue of L. dispar larvae, and in the whole midgut and midgut tissue of E. chrysorrhoea larvae. Different expression patterns were detected for enzyme isoforms in tissues of larvae exposed to dietary fluoranthene. Total GSH content and GST activity increased in E. chrysorrhoea larval midgut tissue. Significantly decreased SOD activity in hemolymph of L. dispar larvae, and opposite changes in CAT activity were recorded in the hemolymph of larvae of two insect species. The tissue-specific responses of enzymes to dietary fluoranthene, recorded in each species, enabled the larvae to overcome the pollutant induced oxidative stress, and suggest further assessment of their possible use as early-warning signals of environmental pollution.