Search

Silkworm (Bombyx mori) pupa protein (SPP) is a high-quality source of animal protein with substantial nutritional benefits and health value. To develop an efficient extraction method for SPP that is environmentally friendly, we selected choline hydroxide ionic liquid (CH-IL) as the extraction solvent and performed orthogonal experiments to optimize the extraction conditions. We demonstrated that 3% CH-IL, a solid-to-liquid ratio (g/mL) of 1:30, an extraction temperature of 40 °C, and an extraction time of 1 h facilitated the most efficient extraction. Compared to the conventional alkali solubilization–acid precipitation method, the CH-IL extraction increased protein content by 12.14%. Protein structure analysis showed that the β-sheet content increased by 10.98% and that of disulfide bonds reduced by 16.4%. The processing properties of the CH-IL extracted protein showed that the solubility, emulsification, and foaming capacity were enhanced by 82.87%, 15.44%, and 18.97%, respectively. The physical properties of SPP remarkably improved relative to the increased stretching of the polypeptide chains. The findings of this study provide technical knowledge that will enhance the processing performance of pupal proteins.

Anthocyanin (cyanidin-3-O-glucose) is a natural water-soluble pigment with a robust antioxidant capacity. However, its poor stability and bioavailability limits its application as a functional food ingredient. This study explored the ability of the silkworm pupa protein-glucose (Spp-Glu) conjugate, developed under wet-heating conditions, to improve the thermal stability and antioxidant activity of cyanidin-3-O-glucose (C3G) at pH 3.0 and 6.8. The characterization experiments suggested that C3G complexed with the Spp-Glu conjugate could modify the protein's microenvironment and cause unfolding of the protein's secondary structures under varied pH conditions. Spectroscopic techniques further revealed the formation of complexes via hydrophobic interactions and static quenching processes when C3G was bound to Spp or Spp-Glu. The formation of these complexes effectively attenuated C3G degradation, thereby enhancing its stability under heat treatment over a range of pH values, and the experiments measuring antioxidant activity suggested that the Spp-Glu conjugate formed does not affect the efficacy of C3G after complexation. Therefore, our study suggests that Spp-Glu has the potential to effectively protect and deliver anthocyanins during industrial application for functional food formulation.

In this study, we isolated and characterized an immunomodulatory peptide from silkworm (Bombyx mori) pupa protein hydrolysates. Ultrasound-pretreated hydrolysates were prepared by alcalase digestion and their pro-proliferative activity was assessed with the splenic lymphocyte proliferation assay. Peptide fractions exhibiting the highest activity were purified by Sephadex-G100 and -G15 gel filtration chromatography. The structure of the purified peptides was analyzed using Liquid Chromatography Electrospray Ionisation Tandem Mass Spectrometry (LC–ESI–MS/MS); their digestive stability and their effects of the expression on immune-related cytokines were also evaluated. We purified a novel immunomodulatory peptide with a molecular weight of about 441.06 Da. The amino acid sequence was Asp-His-Ala-Val (DHAV). The splenocyte proliferation rate was 91.1% (P

Major royal jelly proteins (MRJPs) are important protein components of bee royal jelly (RJ) and exhibit various biological and pharmacological activities. The antimicrobial activities of the royalisin and the jelleines contained within MRJP 1 and MRJP 2 in RJ have been elucidated. However, the antimicrobial effects of other MRJPs remain largely unknown. In this study, we demonstrated the antimicrobial activity of the Asiatic honeybee (Apis cerana) MRJP 4 (AcMRJP4). Recombinant AcMRJP4 was expressed as a 63-kDa protein in baculovirus-infected insect cells. We examined the antimicrobial activity of recombinant AcMRJP4 against bacteria, fungi, and yeast. The mechanisms underlying the antimicrobial activity of AcMRJP4 were assessed using western blot analysis, immunofluorescence staining, and scanning electron microscopy. Recombinant AcMRJP4 bound to the cell walls of bacteria, fungi, and yeast and induced structural damage in the microbial cell walls. AcMRJP4 has an antimicrobial role and exhibits a broad spectrum of antimicrobial activities against bacteria, fungi, and yeast. We demonstrated that AcMRJP4 functions as an antimicrobial agent with activity against bacteria, fungi, and yeast. Together, our data identified a novel function of MRJP 4 as an antimicrobial agent.

The entomopathogenic mushroom Cordyceps militaris is a storehouse of various medicinal compounds and pharmacological effects. However, the high frequency of strain degeneration during subculture and preservation severely limits the large-scale production of C. militaris. DNA methylation is an important epigenomic modification involved in gene regulation. In this study, we used bisulfite sequencing for DNA methylation profiling of wild-type and mutant C. militaris. The differentially methylated regions (DMRs) of the two types were analyzed using Gene Ontology (GO) clustering and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. DNA methylation levels of the wild-type and mutant-type C. militaris were 0.48% and 0.56%, respectively. Methylation appeared at CHH dinucleotides in 58.62% and 58.20% of all methylated cytosine sites in the wild and mutant types, respectively. In all, 188 DMRs were identified from the wild and mutant types. Most of the DMRs ranged from 200 to 350 bp in length. KEGG pathways of the expression of DMR-related genes, which are involved in pyruvate metabolism, glycerophospholipid metabolism, DNA replication, and N-glycan biosynthesis. This contributes to the knowledge and understanding of the possible mechanisms of C. militaris strain degeneration.

In this paper, the interaction of silkworm pupae protein (SPP) with cyanidin-3-O-glucoside (C3G) was studied the protective anthocyanins stability. Characterization experiments suggested that C3G-SPP complexes mainly through hydrophobic interactions, with a decrease in the α-helix content and increases in the β-sheet and β-turn contents. Fluorescence results revealed that C3G quenched the intrinsic fluorescence of SPP by static quenching. The highest quenching constant, Kq, was recorded to be1.26 × 1012 M−1s−1 for the SPP preheated at 80 °C. Following the C3G-SPP complexes, the degradation rate constant decreased, and the half-life of C3G was prolonged from 64.81 ± 1.07 to 261.99 ± 13.32 min at 80 °C (p

The chemical composition and pharmacological effects of Cordyceps militaris are similar to those of Cordyceps sinensis, with the former undergoing greater development and utilization. Strain degeneration is a common phenomenon that occurs with high frequency during the subculturing of C. militaris, however, and the mechanism underlying strain degeneration remains unclear. In this study, we used touch-down PCR to compare the ITS1 + 5.8S + ITS2, 18S, 28S and mating-type (MAT) regions sequence of wild-type and degenerated strains of C. militaris. We also used quantitative real-time PCR to analyze expression levels of the CmMAT gene. Sequence analysis showed that the ITS1 + 5.8S + ITS2 and 28S regions of degenerated and wild-type strains were completely identical, the 18S region of the degenerated strain contained seven single-base mutations, including six base substitutions and one single-base insertion. Compared with the wild-type strain, the degenerated strain contained a deletion of the MAT1–2-1 region, three base substitutions in the MAT1–1-1 region, and a base substitution in the MAT1–1-2 region that causes a glycine-to-valine amino acid substitution. Quantitative real-time PCR analysis detected no CmMAT1–2-1 gene expression in the degenerated strain, confirming the deletion of the CmMAT1–2-1 gene. Expression levels of the CmMAT1–1-1 and CmMAT1–1-2 genes were significantly down-regulated to only 7.5 % and 4.4 %, respectively, that of the wild-type strain. These results indicate that 18S and MAT region mutations, as well as down-regulated of CmMAT gene expression levels, may play important roles in C. militaris degeneration. This study provides a theoretical basis for further elucidation of the molecular mechanisms of C. militaris degeneration.

To improve the properties of silkworm ( Bombyx mori ) pupa protein (SPP), the effects of different modification techniques included enzymolysis, ultrasound, and micronization on the molecular structure and bioactivity of SPP has been evaluated in this study. The results showed that enzymolysis modification led to the most substantial change in the area and shape of the infrared (IR) absorption peaks, decreased in size with an increased number of cracks of modified protein by scanning electron microscopy images, followed by ultrasound and micronization modification. After modification by enzymolysis, ultrasound, and micronization, the sulfhydryl concentrations of SPP were increased by 48.56%, 34.82%, and 12.46%, respectively; disulfide bond concentrations were reduced by 40.47%, 10.79%, and 11.72%, respectively. Furthermore, the ratios of essential amino acids among total amino acids increased by 13.85%, 2.22%, and 6.93%, respectively. Antioxidant activity and ACE-inhibitory activity of SPP also increased notably after modifications.

The silkworm, Bombyx mori (B. mori) is an important economic insect which ingests mulberry leaves and products the silk in industry. Chlorfenapyr is a new halogenated pyrrole insecticide which has been promoted for the control of mulberry insect pests in China. However, the detoxification mechanism of the silkworm to chlorfenapyr has not been investigated yet. In the present study, we first estimated the LC30 dose of chlorfenapyr for 3rd instar B. mori larvae, and then, in order to characterise the chlorfenapyr detoxification mechanism, the transcriptomes of chlorfenapyr-treated and untreated 3rd instar B. mori larvae were compared using RNA-sequencing. In total, 146, 533, 126 and 148, 957, 676 clean reads were obtained from insecticide-treated and control silkworm larvae, respectively, and these reads generated 10, 954 genes. The transcriptional profile of silkworm larvae was significantly influenced by chlorfenapyr treatment. A total of 1196 differentially expressed genes (DEGs) were identified in insecticide-treated and control B. mori larvae, in which 644 genes were upregulated and 552 genes were downregulated. Results showed that multiple DEGs were enriched in detoxication-related gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Eleven detoxifying enzyme genes which differentially expressed were screened, and their expression patterns were validated by qRT-PCR. Furthermore, we successfully knocked down all differentially upregulated detoxifying enzyme genes, and a bioassay showed that the mortality of chlorfenapyr-treated silkworm larvae was significantly higher after silencing these genes than in groups injected with dsGFP. The present study reveals the molecular basis of silkworm detoxification to chlorfenapyr exposure, and provides new insights into the management of insecticide damage in the silkworm.

Delta 6-fatty acid desaturase is a membrane-bound enzyme, which is the rate-limiting factor in the biosynthesis of polyunsaturated fatty acids. In this study, a novel delta 6-desaturase gene was cloned from Bombyx mori (BmD6DES). Sequencing analysis revealed that BmD6DES has an open reading frame of 1357 bp that encodes 448 amino acids. Heterologous expression in yeast demonstrated that BmD6DES could synthesize γ-linolenic acid (GLA, 18:3, Δ6,9,12) by utilizing the endogenous substrate linoleic acid (LA, 18:2, Δ9,12). We found that BmD6DES transcripts were distributed in almost all B. mori tissues, with high expression levels observed at the 5th instar larval, pupal, and adult moth stages. A functional analysis of BmD6DES was performed by measuring mRNA levels after temperature stress, fungal infection, and RNA interference (RNAi). The results indicated that the highest expression of BmD6DES was observed at low temperatures (0 °C) and 6 h to 36 h after fungal infection. qPCR analysis demonstrated that BmD6DES mRNA levels in pupa after BmD6DES RNAi treatment were significantly reduced from 12 h to 72 h compared to those in the control group. Our findings suggest that BmD6DES not only induces the formation of the third carbon–carbon double bond in the LA carbon chain, but also leads to sensitivity to low-temperature stress and fungal infection. These results imply that BmD6DES is a key gene in the γ-linolenic acid pathway during B. mori development.

In this paper, we examined the use of 1-methylcyclopropene (1-MCP) and ClO2 as safe and effective preservative agents for mulberries. Individual treatment of 1 g/m3 1-MCP or ClO2 at 20 g/m3 significantly delayed fruit senescence and did not affect sensory qualities. Combined treatment of 1 g/m3 1-MCP and 12 g/m3 ClO2 yielded the lowest mildew rate, and the fruits remained plump and bright with bright green stalks on the third day. In contrast, most of the mulberry fruits of the control group were moldy on the second day, with no edible value. Physiological quality studies showed that combined treatment of 1-MCP and ClO2 could effectively maintain the nutritive qualities, such as ascorbic acid and polysaccharide contents and titratable acidity, and delay malondialdehyde accumulation. Thus, combined treatment of 1-MCP and ClO2 prolonged the shelf-life of mulberries for about 3 days. Our finding is of great value to the horticulture and fruit postharvest. Practical Applications Current methods used for fruit and vegetable preservation might leave harmful chemical residues. 1-Methylcyclopropene (1-MCP) has been found to effectively regulate fruit maturation and prolong fruit shelf-life, and ClO2 is a strong oxidant that leaves no harmful substances or odor residuals after treatment. We show that combined treatment of 1 g/m3 1-MCP and 12 g/m3 ClO2 can maintain the mulberry fruit's exterior qualities and significantly reduce the rotting rate and weight loss ratio. We demonstrate the applicability of the combined treatment of (1-MCP) and ClO2 to prolong the shelf-life of mulberries for about 3 days. Our finding is of great value to the horticulture and postharvest and industry because mulberry fruit has a very low shelf-life. Its maturation period is extremely short, rendering its storage, transportation, and sale very difficult.

Insect cuticular melanization is regulated by the prophenoloxidase (proPO)-activating system, which is also involved in the innate immune reaction. Here, we demonstrate how the differentiation of the proPO-activating system is regulated toward a cuticular melanization or innate immunity function in silkworm ( Bombyx mori ) pupae. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating enzyme and proPOs, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism that is mediated by the priming of the proPO system.

Although many studies have shown the antitumor properties of Cordyceps militaris (artificial cultivated from Bombyx mori pupa) polysaccharides, little is known regarding the mechanism of its effects. This study was conducted to determine the mechanism of antitumor effects of C. militaris polysaccharide extract by evaluating apoptosis rate and cell cycle progression status in human liver cancer cell SMMC-7721, stomach cancer cell BGC-823 and breast cancer cell MCF-7. Results showed that C. militaris polysaccharides inhibited proliferation of SMMC-7721, BGC-823 and MCF-7 cells with an IC 50 of 192 ± 23.2 μg/mL, 237 ± 12.7 μg/mL and 165 ± 16.3 μg/mL, respectively. We also found that C. militaris polysaccharides at increasing concentrations induced apoptosis dose dependently in those cancer cells: apoptosis rates were 48.3%, 59.4% and 70.9% for SMMC-7721, 41.3% and 57.0%, 72.2% for BGC-823 and 61.3%, 66.9% and 80.6% for MCF-7 at 110, 156 and 323 mg/mL of C. militaris polysaccharides, respectively. C. militaris polysaccharides arrested SMMC-7721, BGC-823 and MCF-7 cells at G 0 /G 1 and G 2 /M phases with corresponding decrease in S-phase. This study suggests that C. militaris polysaccharides may exert its antitumor effects in those cancer cells by suppressing its growth, arresting the G 0 /G 1 -phase, reducing DNA synthesis and inducing apoptosis.

Silkworm pupa ( Bombyx mori ) protein (SPP) was treated by ultrasound, and then was hydrolyzed using alcalase. The hydrolysate with the highest ACE inhibitory activity was obtained at hydrolysis of 50 min when SPP was treated at power of 410 W/100 ml for 32 min. The hydrolysate was fractionated by ultrafiltration, and peptide with the highest ACE inhibitory activity was purified from 50 = 12.82 μM), and it was stable against the gastrointestinal proteases. The molecular docking study revealed that ACE inhibitory activity of the tripeptide was mainly attributed to the hydrogen bond interactions and Zn(II) interaction between the tripeptide and ACE. These results suggest that the tripeptide is a potential natural ACE inhibitor that can be used as drug or functional food ingredient.

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

Bombyx mori cathepsin B (BmCatB) is involved in the programmed cell death of the fat body during B. mori metamorphosis. For a better understanding of the functional regulatory mechanism, the promoter region of BmCatB in the transcriptional regulation has been identified and analyzed in the present study. BmCatB promoter region performed by the 5' truncation or mutagenesis of EcREs was inserted in the pFA3Luc-A3RL double fluorescence expression vector to activate the fireflies luciferase (FLuc) gene. The results indicated that the dual-luciferase activity of BmCatB gene in the silkworm larval fat body is regulated by the length of promoter. Site-directed mutagenesis of EcRE experiment has shown that the EcREs are up-regulated significantly in the regulation of the BmCatB promoter. A 142bp region (-1165 to -1023) and EcREs are the mainly fat-body tissue-specificity related region and could function as a core promoter element.

While screening for cellulase-producing fungi from insect gut, a fungus with high endoglucanase (carboxymethyl cellulase; CMCase) activity was isolated from the larval gut of Bombyx mori. Based on morphological characteristics and using an 18S rRNA-based molecular phylogenetic approach, the fungus, strain BMC-2, was identified as a Mucor sp. expressing a novel alkalotolerant cellulase. The maximum production of cellulase by the BMC-2 strain was observed at 55 °C and pH 8.0. The CMCase activity was inhibited by Cu2 + > Na+ > Zn2 + > Mg2 + > Ba2 +, and induced by Ca2 +, Mn2 +, Fe2 +, and K+.

Insect growth-blocking peptides (GBPs) exhibit growth-blocking and paralytic activity. Low concentrations of GBP stimulate larval growth, whereas high concentrations of GBP significantly retard larval growth. Here, we show that morphological abnormalities and lethality were induced in silkworm ( Bombyx mori ) larvae by high concentrations of GBP. Active B. mori GBP (BmGBP) was produced by treating recombinant proBmGBP (expressed in baculovirus-infected insect cells) with bovine factor Xa. When silkworm larvae on day 1 of the fifth-instar stage were injected between the seventh and eight abdominal segments with BmGBP (100 or 500 ng/larva), the larval–pupal and pupal–adult transformations of these silkworms were delayed in a dose-dependent manner. However, a high concentration (2000 ng/larva) of BmGBP or Spodoptera exigua GBP (SeGBP) acutely induced morphological abnormalities and death in silkworm larvae. In silkworm larvae treated with high concentrations of GBPs, the ingested food excessively accumulated in the foregut, which caused extreme swelling in both the thorax and the foregut and resulted in larval death. Therefore, these results not only provide insight into the effect of insect GBPs on gut physiology but also reveal a novel function of insect GBPs.

The gene encoding cathepsin D of silkworm, Bombyx mori (BmCatD) is specifically expressed in the larval fat body and pupal gut, and plays an important role in the programmed cell death during metamorphosis. To identify element involved in this transcription-dependent spatial restriction, truncation and deletion of the 5' terminal from the BmCatD promoter were conducted in vivo. The recombinant AcMNPV vector (Autographa californica multiple nucleopolyhedrovirus) with a dual-luciferase quantitative assay system was used as the transfer. A 289 bp DNA sequence (-1,214 to -925) upstream of the transcriptional start site is found to be responsible for promoting tissue-specific transcription. Further analysis of a series of deletion within the 289 bp region of overlapping deletion showed that a 33 bp region (-1,071 to -1,038) sequence suppresses the ectopic expression of the BmCatD promoter. These results suggest that this 33 bp region could function as a promoter element in the tissue-specificity expression.

Cathepsins are well-characterized proteases that are ubiquitously expressed in lysosomes. Previous work revealed that Bombyx mori cathepsins B and D are expressed in the fat body and undergo decomposition during larval-pupal metamorphosis. Quantitative RTPCR was performed to detect cathepsin gene expression at the transcription level when challenged by B. mori nuclear polyhedrosis virus (BmNPV), temperature and hormones (20-hydroxyecdysone (20E) and juvenile hormone analogue (JHA)). mRNAs encoding cathepsins B and D were significantly enhanced after the larvae were infected with BmNPV, and the peak of the induction appeared at 1 day before spinning. This attenuated the inducing effect on cathepsin expression caused by infection. Temperature shock induced cathepsin expression at the later stage of the 5th instar, and transcription levels varied with development stage and temperature. Cathepsin B and D mRNA expression in the fat body were significantly induced by JHA at the day before spinning, and with 20E, the expression reached a peak at the last day of the 5th instar. Cathepsin B and D mRNA expression exhibited detectable changes post-treatment, without significant differences between or among the hormone concentrations.

The polysaccharide fraction, CM-hs-CPS2, was isolated from fruiting bodies of culturedCordyceps militaris grown on solid rice medium by hot water extraction and ethanol precipitation, and purified by ion exchange column (DEAE-cellulose-52) and gel filtration column (Sephadex G-100) chromatography. Its structural characterizations were analyzed by gas chromatograph (GC) and fourier transform infrared spectroscopy (FT-IR). Results indicated that CM-hs-CPS2 was a kind of sulfating acidic polysaccharide containing acetamido group. The monosaccharide composition analysis showed that CM-hs-CPS2 was composed mainly of mannose, glucose and galactose in a molar ration of 1.35: 8.34: 1.00, and linked by α-glycosidic linkage. The studies on antioxidant activities of CM-hs-CPS2 were evaluated by various methods in vitro. Results showed that DPPH· scavenging activity, reducing power and ferrous ion chelating activity of CM-hs-CPS2 (8 mg/ml) were 89%, 1.188 and 85%, respectively. Key words: Cordyceps militaris, polysaccharide, structure, bioactivity.

Glutathione S-transferases (EC 2.5.1.18; GSTs) are multifunctional enzymes that are mainly involved in xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTS gene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H(2)O(2) overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H(2)O(2) overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3h after H(2)O(2) exposure, whereas BiGSTS and BiGSTD are highly induced at 9h after H(2)O(2) exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitus are differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC 1.11.1.12) is an antioxidant enzyme that reduces lipid hydroperoxides in biomembranes. Here, we cloned and characterized cys-PHGPx from the bumblebee Bombus ignitus (Bi-PHGPx). The Bi-PHGPx gene consists of 4 exons, encoding 168 amino acid residues with a canonical cys-codon at residue 45 and active site residues Gln(82) and Trp(134). Recombinant Bi-PHGPx, expressed as a 19 kDa protein in baculovirus-infected insect cells, exhibited enzymatic activity against PLPC-OOH and H(2)O(2) using glutathione as an electron donor. Tissue distribution analyses showed the presence of Bi-PHGPx in all tissues examined. Bi-PHGPx transcripts were upregulated by stresses, such as wounding, H(2)O(2) exposure, external temperature shock, and starvation. Under H(2)O(2) overload, the RNA interference (RNAi)-induced thioredoxin peroxidase (BiTPx1)-knock-down B. ignitus worker bees showed upregulated expression of Bi-PHGPx in the fat body. These results indicate that Bi-PHGPx is a stress-inducible antioxidant enzyme that acts on phospholipid hydroperoxide and H(2)O(2).

Glutathione S-transferase (GST) is a gene family generally associated with detoxification and plays an important role in detoxifying exogenous compounds. The silkworm Bombyx mori is an important economic animal for silk production. However, it is liable to infection by a number of viruses and chemical agents that can contaminate its food and growing environment. Here we conducted a comparative study of strain- and tissue-specific expressions of GST in the silkworm under infections by Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori densonucleosis virus (BmDNV) and under the stress of pesticides (insecticide and herbicide). BmDNV induced an increase of GST at 24 h and a decrease at 48 and 72 h in the midgut of the DNV-susceptible strain and a decrease at 24 h and increase at 48 and 72 h in the midgut of the DNV-tolerant strain. BmDNV induced a significant increase of GST from 24 to 72 h in the fat body of both DNV-susceptible and DNV-tolerant strains. In contrast, BmNPV induced a significant decrease of GST in both the fat body and midgut of the NPV-susceptible strain and induced increase of GST from 24 to 48 h in the midgut and at 72 h in the fat body of the NPV-tolerant strain. All of these results suggest that BmGST activity varies with the strain, tissue, feeding behavior, and developmental stage of the silkworm. On treating silkworm larvae with pesticides (insecticide and herbicide), expression of the BmGSTS2 gene increased noticeably in the midgut and reached a peak at 6 to 12 h. The mRNA level of BmGSTS2 in the midgut increased during administration of the chemicals, suggesting that the induction of BmGSTS2 is part of the defense mechanism against exogenous chemicals.

Three polysaccharides, PSPP1-1, PSPP2-1 and PSPP3-1, were isolated from purple sweet potato. The three polysaccharides belonged to β-type polysaccharides and contained low proportions of proteins and uronic acids. PSPP1-1 and PSPP3-1 with molecular weights of 33.3 and 75.3 kDa, respectively, were composed of rhamnose, xylose, glucose and galactose, whereas PSPP2-1 with molecular weight of 17.8 kDa was composed of rhamnose and galactose. The three polysaccharides possessed in vitro antioxidant (scavenging DPPH radicals, chelating ferrous ions and reducing power) and antitumor activities (against SGC7901 and SW620 cells) in a dose-dependent manner. Among the three polysaccharides, PSPP2-1 exhibited the strongest reducing power, scavenging activity on DPPH radicals and chelating capability on ferrous ions. PSPP1-1 showed the strongest inhibitory activities on the growth of SGC7901 and SW620 cells. In addition, flow cytometry results showed that PSPP1-1 could induce apoptosis in SGC7901 and SW620 cells. These results suggest that polysaccharides from purple sweet potato are potential natural antioxidant and antitumor agents that can be used as drugs or functional food ingredients.

A digestive beta-glucosidase cDNA was cloned from the silkworm, Bombyx mori. The B. mori beta-glucosidase cDNA contains an open reading frame of 1473 bp encoding 491 amino acid residues. The B. mori beta-glucosidase possesses the amino acid residues involved in catalysis and substrate binding conserved in glycosyl hydrolase family 1. Southern blot analysis of genomic DNA suggested the B. mori beta-glucosidase to be a single gene. Northern blot analysis of B. mori beta-glucosidase gene confirmed larval midgut-specific expression. The B. mori beta-glucosidase mRNA expression in larval midgut was detectable only during feeding period, whereas its expression was downregulated during starvation. The B. mori beta-glucosidase cDNA was expressed as a 57-kDa polypeptide in baculovirus-infected insect Sf9 cells, and the recombinant beta-glucosidase was active on cellobiose and lactose, but not active on salicin, indicating that the B. mori beta-glucosidase possesses the characteristics of the Class 2 enzyme. The enzyme activity of the purified recombinant beta-glucosidase expressed in baculovirus-infected insect cells was approximately 665 U per microg of recombinant B. mori beta-glucosidase. The purified recombinant B. mori beta-glucosidase showed the highest activity at 35 degrees C and pH 6.0, and were stable at 50 degrees C at least for 10 min. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that the recombinant B. mori beta-glucosidase is N-glycosylated, but the carbohydrate moieties are not essential for enzyme activity.

We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family 45 cloned from the mulberry longicorn beetle, Apriona germari (Ag-EGase I), is composed of 237 amino acid residues and has a potential N-glycosylation site at 97-100 amino acid residues (NSTF). We here describe the N-glycosylation and its role for enzymatic activity of the Ag-EGase I. The N-glycosylation of Ag-EGase I was revealed by the treatment of tunicamycin to the recombinant virus-infected insect Sf9 cells and by endoglycosidase F to the purified recombinant Ag-EGase I, demonstrating that the carbohydrate moieties are not necessary for secretion but essential for Ag-EGase I enzyme activity. To further elucidate the functional role of the N-glycosylation in Ag-EGase I, we have assayed the cellulase enzyme activity in Thr99Gln mutant. Lack of N-glycosylation in Ag-EGase I showed no substantial enzyme activity. This result demonstrates that N-glycosylation at site 97-100 amino acid residues (NSTF) is essential for enzyme activity.

Background: Mulberry fruits are a superior source of polyphenol, especially anthocyanins that contribute potentially to the beneficial effects which include reducing the risk of cardiovascular diseases and cancers with antioxidant, anti-inflammatory, and chemoprotective properties. Objectives: In this study, purification of the polyphenol-rich extract from mulberry fruit (MPE) was purified and assessed the activities of antioxidant and hemolysis protective in vivo and in vitro. Materials and Methods: Antioxidant activities in vitro was measured by quantifying its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, reducing power and Fe2+-chelating ability. MPE was purified by high-pressure liquid chromatography (HPLC) and analyzed individual polyphenols using liquid chromatography–mass spectrometry (LC-MS)/MS. Results: The total polyphenol content was 147.69 ± 0.02 mg gallic acid equivalents (GAE)/g dried weight (DW) in the extract and 403.55 ± 0.02 mg GAE/g DW in the purified extract. Further identification by HPLC-ultraviolet-visible and LC-MS/MS analysis indicated in MPE, an anthocyanin compound, cyanidin-3-O-glucoside. With regard to in vitro assays, MPE possessed antioxidant effect, especially in Fe2+ chelating ability with an IC50 value of 1.016 mg/mL. The protective effects on mouse red blood cell hemolysis and lipid peroxidation ex vivo were dose and time dependent. Conclusion: It indicates that MPE could be a good candidate for future biomedical applications to promote human health with limited side effects. SUMMARY Mulberry fruit is an excellent source of polyphenols, in particular, anthocyanins, which has infinite health benefits. This study determined the predominant anthocyanin, cyanidin-3-glucoside, could possibly be the rationale behind the antioxidant and antihemolytic effect of MPE. Results indicate that MPE could be a good candidate for future biomedical applications to promote human health with limited side effects. Abbreviations used: MPE: Purification of the polyphenol-rich extract from mulberry fruit, LC-MS: Liquid chromatography–mass spectrometry, HPLC: High-pressure liquid chromatography, DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging activity, RBC: Red blood cell, GAE: Gallic acid equivalent, FeCl2: Ferrous chloride, H2O2: Hydrogen peroxide, EDTA-2Na: Ethylenediaminetetraacetic acid disodium salt, PBS: Phosphate-buffered saline, TCA: Trichloroacetic acid, TBA: 2-thiobarbituric acid, FeSO4: Ferrous sulphate, MDA: Malondialdehyde, VC: Vitamin C, DW: Dried weight.

Silkworm pupa ( Bombyx mori ) protein was hydrolyzed using gastrointestinal endopeptidases (pepsin, trypsin and α-chymotrypsin). Then, the hydrolysate was purified sequentially by ultrafiltration, gel filtration chromatography and RP-HPLC. A novel ACE inhibitory peptide, Ala-Ser-Leu, with the IC 50 value of 102.15 μM, was identified by IT-MS/MS. This is the first report of Ala-Ser-Leu from natural protein. Lineweaver–Burk plots suggest that the peptide is a competitive inhibitor against ACE. The molecular docking studies revealed that the ACE inhibition of Ala-Ser-Leu is mainly attributed to forming very strong hydrogen bonds with the S1 pocket (Ala354) and the S2 pocket (Gln281 and His353). The results indicate that silkworm pupa ( B. mori ) protein or its gastrointestinal protease hydrolysate could be used as a functional ingredient in auxiliary therapeutic foods against hypertension.

An antimicrobial endophytic bacterium strain, ME-2, was isolated from fresh mulberry twig tissues, and was identified. In addition, antimicrobial activities of the isolated strain were evaluated against Bacillus thuringiensis, Escherichia coli, Staphylococcus aureus,Beauveria bassiana and Mycoid bacillus using the antagonism test method. The results showed that, strain ME-2 was gram-positive, aerobic, motile, nitrate-reduction-positive, spore-formation-positive, methyl-red-negative, 0.6-0.7 × 1.5-1.6 μm long and rods. The strain could utilize D-glucose, D-lactose, sucrose, starch, citrate, gelatin and casein, and it was slightly halophilic with growth occurring at 10% (w/v) NaCl. According to the morphological, biochemical and physiological properties and sequence analysis of 16S rRNA gene, the strain was identified as Bacillus subtili (GeneBank accession No. JQ900623). Moreover, the results also showed that strain ME-2 exhibited strong antimicrobial activities against B. thuringiensis (76.0%), E. coli (69.8%), S. aureus(45.8%), B. bassiana (64.2%) and M. bacillus (86.3%). It was concluded that, strain ME-2 and its antimicrobial materials might be considered as a potential antimicrobial agents in the food and pharmaceutical industries. Key words: Antimicrobial endophytic bacterium, identification, biochemical and physiological properties, 16S rRNA gene, antimicrobial activity.

Bombyx mori Cathepsin D (BmCatD) is specifically expressed in the fat body, and plays a critical role for the programmed cell death of the larval fat body and pupal gut during metamorphosis. To better understand the transcriptional control of BmCatD expression, we conducted this study to identify the ecdysone response elements (EcREs) in the BmCatD promoter and clarify their regulational functions. We inserted EcREs into a recombinant AcMNPV (Autographa californica multiple nucleopolyhedrovirus) vector and performed luciferase assay with a dual-luciferase quantitative assay system. Three putative EcREs were located at positions −109 to −99, −836 to −826 and −856 to −846 relative to the transcription start site. Overlapping deletion studies of this EcRE region showed that the three EcREs could suppress the ectopic expression of the BmCatD promoter. EcRE mutations resulted in the loss of the fat body-specific expression of the BmCatD gene. These results suggest that the EcREs are vital for activation of the promoter by 20-hydroxyecdysone (20E) in the larval fat body and further support the crucial role of ecdysone signaling to control cathepsin D gene transcription. It may suggest that the heterodimeric complex EcR/USP mediates the activation of ecdysone-dependent BmCatD transcription in the larval fat body of B. mori.

Two acidic polysaccharide fractions, CM-jd-CPS2 and CM-jd(Y)-CPS2, were isolated from the fruiting bodies of cultured Cordyceps militaris grown on solid rice medium and silkworm pupa, respectively, by hot-water extraction, ethanol precipitation and fractionation using ion-exchange column (DEAE-cellulose-52) and gel-filtration column (Sephadex G-100) chromatography. Their structural characterizations were performed by gas chromatography and fourier-transform infrared spectroscopy. Some differences existed between their structures, which indicated that culture media could influence the structure of polysaccharides of C. militaris. The antioxidant activities of CM-jd-CPS2 and CM-jd(Y)-CPS2 were evaluated by various methods in vitro. They had strong 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity and ferrous ion-chelating capacity, but moderate reducing power. The antioxidant activities of CM-jd(Y)-CPS2 were slightly higher than those of CM-jd-CPS2. These two acidic fractions were evaluated for proliferation of mouse splenocyte activity in vitro. They both possessed does-dependent mitogenic effects on mouse splenocytes, and could synergistically promote murine T- and B-lymphocytes induced by Con A and LPS. CM-jd(Y)-CPS2 exhibited stronger stimulatory activities upon immunomodulation than CM-jd-CPS2. These results are beneficial for the interpretation of the connection between polysaccharide structures and their biological activities.

Proteolytic enzymes are involved in insect molting and metamorphosis, and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compare the expression profiles of B. mori cathepsins B (BmCatB) and D (BmCatD) during normal development and after RNA interference (RNAi)-mediated inhibition. BmCatB is induced by 20-OH-ecdysone, and is expressed in the fat body of B. mori during molting and the larval-pupal and pupal-adult transformations, where its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval-pupal transformation, and BmCatB RNAi treatment resulted in an arrest of the larval-pupal transformation. RNAi-mediated BmCatB knockdown sustained the expression of BmCatD during the larval-pupal transformation. On the other hand, when BmCatD was inhibited via RNAi, the expression of BmCatB was upregulated. Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis, and that BmCatB and BmCatD contribute to B. mori metamorphosis.

Background: Many plants possess antioxidants that exhibit additive or synergistic activities. Objective: In this study, an ethanol-extracted flavonoid extracted from mulberry fruit (FEM) was evaluated for the antioxidant activity in vitro and the hemolysis in red blood cell (RBC) and lipid peroxidation in liver in vivo. Materials and Methods: Antioxidant activities in vitro were measured by quantifying its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity, reducing power, and Fe2+-chelating ability. FEM inhibits hemolysis in RBCs and effects of lipid peroxidation in the liver were estimated. Results: The total content of flavonoid compounds was 187.23 mg of quercetin equivalents per grams dried material. In the in vitro assays, FEM demonstrated a strong antioxidant effect, especially in DPPH scavenging activity and reducing power. Mouse RBC hemolysis induced by H2O2 was significantly inhibited by FEM in a dose- and time-dependent manner. The effects of FEM on lipid peroxidation in liver, mitochondria, and microsome were investigated. The percentage of inhibition at high concentration (100 μg/mL) of FEM was 45.51%, 39.36%, and 42.78% for liver, mitochondria, and microsomes, respectively. These results suggest that the FEM possesses a strong antioxidant activity both in vivo and in vitro. SUMMARY The total content of flavonoid compounds in mulberry fruit was 187.23 mg/g dried materialFEM showed a strong antioxidant effect, especially in 2,2-diphenyl-1-picrylhydrazyl scavenging activity and reducing powerMouse red blood cell hemolysis induced by H2O2 was significantly inhibited by FEM in a dose- and time-dependent mannerThe inhibition percentage at high concentration of FEM was 45.51%, 39.36%, and 42.78% for mouse’s liver, mitochondrial, and microsomes, respectively. Abbreviations used: FEM: Flavonoid Extracted from Mulberry fruit, H2O2: Hydrogen peroxide, DPPH: 2,2-diphenyl-1-picrylhydrazyl, EDTA: Ethylene diamine tetraacetic acid, MDA: malondialdehyde, TBA: 2-thiobarbituric acid, RBC: Red blood cells, DNJ: 1-deoxynojirimycin, LDL: low density lipoprotein, ROS: reactive oxygen species, EDTA2Na: Ethylenediaminetetraacetic acid disodium salt.

A novel endogenous β-1,4-endoglucanase (Ag-EGase III) gene belonging to the glycoside hydrolase family (GHF) 5 was cloned from the mulberry longicorn beetle, Apriona germari . The Ag-EGase III gene spans 1061 bp and consists of a single exon coding for 325 amino acid residues. The Ag-EGase III showed 89% protein sequence identity to another beetle, Psacothea hilaris , cellulase belonging to GHF 5. The Ag-EGase III has the potential proton donor and nucleophile amino acids conserved in GHF 5 and two putative N -glycosylation sites. Northern blot and Western blot analyses showed that Ag-EGases were expressed in the gut; Ag-EGase III and Ag-EGase I were expressed in three gut regions, and no Ag-EGase II was found in hindgut, indicating that the foregut and midgut are the prime sites for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase III was expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase III was approximately 1037 U per mg of recombinant Ag-EGase III. The enzymatic property of the purified recombinant Ag-EGase III showed the highest activity at 55 °C and pH 6.0, and was stable at 60 °C at least for 10 min. In addition, the N -glycosylation of Ag-EGase III was revealed by treatment with tunicamycin of recombinant virus-infected insect Sf9 cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity.

We have previously cloned a cellulase [beta-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Ag-EGase I) belonging to glycoside hydrolase family (GHF) 45 from the mulberry longicorn beetle, Apriona germari. We report here the gene structure, expression and enzyme activity of an additional celluase (Ag-EGase II) from A. germari and also described the gene structure of Ag-EGase I. The Ag-EGase II gene spans 1033 bp and consisted of two introns and three exons coding for 239 amino acid residues. The 2713-bp-long genomic DNA of Ag-EGase I also consisted of two introns and three exons. The Ag-EGase II showed 61% protein sequence identity to Ag-EGase I and 51% to another beetle, Phaedon cochleariae, cellulase, belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase II. The Ag-EGase II has 14 conserved cysteine residues and three putative N-glycosylation sites. Northern blot analysis confirmed midgut-specific expression of Ag-EGase II, suggesting that the midgut is the prime site for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase II was expressed as a 36-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase II was approximately 812 U/mg of recombinant Ag-EGase II. The enzymatic properties of the purified recombinant Ag-EGase II showed the highest activity at 50 degrees C and pH 6.0, and were stable at 60 degrees C at least for 10 min.

Background Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. Results Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. Conclusion Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.