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[Display omitted] • Elevated developmental temperature affects gene expression in bumblebee workers more than in queens and males. • Elevated developmental temperature increases reactive oxygen species and iron concentrations in the hemolymphs of workers. • Elevated developmental temperature increases the gene expression of superoxide dismutase and transferrin in workers. Climate change can negatively impact bumblebee populations, which are crucial pollinators in agricultural systems and natural ecosystems. Bumblebee nest temperatures typically range from 27 to 32 °C, and workers engage in wing fanning to maintain a nest temperature below 32 °C. We investigated the gene expression profile in queen, worker, and male bumblebees (Bombus terrestris) at 27 °C, which is the optimal ambient temperature, and 32 °C, which is the upper-temperature limit requiring wing fanning. We found that a developmental temperature of 32 °C significantly increased the expression of genes related to growth, immune response, antioxidants, and chaperones in workers. Compared to workers reared at 27 °C, those reared at 32 °C exhibited a significant increase in reactive oxygen species (ROS) and iron concentrations in the hemolymph. Additionally, the gene expression of superoxide dismutase (SOD) and transferrin (Tf) was significantly increased in workers at 32 °C. These findings indicate that a developmental temperature of 32 °C in bumblebees, especially workers, leads to an increase in gene expression, including SOD and TF , in response to thermal stress. [ABSTRACT FROM AUTHOR]

[Display omitted] • Apis mellifera major royal jelly protein 8 and 9 (AmMRJP 8 and 9) are venom components. • AmMRJP 8 reduces caspase-3 activity and melittin-induced cell apoptosis. • AmMRJP 8 reduces the production of H 2 O 2 and proinflammatory molecules. • AmMRJP 8 exhibits antiapoptotic activity against melittin-induced cell damage. The dominant protein components of honeybee royal jelly (RJ) are major royal jelly proteins (MRJPs), which exhibit various biological properties. However, the biological basis of why bee venom contains MRJPs and what role MRJPs play in bee venom remains to be elucidated. This study reports the antiapoptotic role of MRJP 8 of Apis mellifera venom (AmMRJP 8) in melittin-treated mammalian cells. Recombinant AmMRJP 8 reduced caspase-3 activity and melittin-induced cell apoptosis. Additionally, recombinant AmMRJP 8 decreased the production levels of H 2 O 2 and proinflammatory molecules. These results indicate that MRJP in bee venom plays a role in cell protection in bee venom-induced inflammatory responses. [ABSTRACT FROM AUTHOR]

The present study aimed to evaluate whether mothers with obesity/central obesity and metabolic syndrome before gestation are at higher risk of insulin administration in gestational diabetes mellitus (GDM) to diminish the burden of insulin use during pregnancy. This was a population-based retrospective cohort study conducted using data from the National Health Information Database of Korea. We identified all deliveries from January 1, 2011 to December 31, 2015 (N = 1,214,655). Among the deliveries, we identified mothers with pre-pregnancy health checkup records and without previous diabetes history (N = 325,208). Hazards of insulin use in GDM were calculated based on pre-pregnancy obesity/central obesity and metabolic syndrome. Hazards of insulin use in GDM increased proportionately with an increase in the pre-pregnancy body mass index (BMI) and waist circumference (WC). After the adjustment for clinical factors, high BMI group (≥30 kg/m2) and high WC group (≥100 cm) were significantly associated with higher hazard ratios (HRs) (HR 4.161, 95% Confidence interval [CI] 3.381–5.121, P < 0.001 and HR 2.563, 95% CI 1.769–3.712, P < 0.001, respectively). The presence of pre-pregnancy metabolic syndrome significantly increased the hazard of insulin use in GDM (0.54% vs. 5.04%). In the presence of obesity (BMI ≥ 25 kg/m2) or central obesity (WC ≥ 85 cm), HRs of insulin use in GDM were 2.637 (95% CI 2.275–3.056) and 1.603 (95% CI 1.023–2.511), respectively, after adjustment for clinical factors. The presence of pre-pregnancy obesity/central obesity and metabolic syndrome in Korean mothers is associated with increased risk of insulin use in GDM. [ABSTRACT FROM AUTHOR]

• Cordyceps militaris extracts (CE) and Paecilomyces tenuipes extracts (PE) improve hyperlipidemia. • CE and PE improve hyperglycemia. • CE and PE regulate expression of genes related to hyperlipidemia and hyperglycemia. Ascocarps of some species of the genus Cordyceps have long been used as a traditional medicine and food source for promoting human health. The compound cordycepin, isolated from C. militaris ascocarps (CE), show similar health effects to CE. In this study, we investigated and compared the anti-obesity and antidiabetic effects of dietary CE and Paecilomyces tenuipes ascocarps (PE) in mice. In addition, we investigated their effects on the expression of genes related to the regulation of obesity and diabetes. We found that dietary CE and PE suppressed body weight gain and fat accumulation in the liver and adipocyte tissues of mice fed a high-fat diet (HFD). Enzyme and lipid profiles induced by HFD returned to normal with CE or PE treatment. Dietary CE or PE reduced fasting blood glucose and serum insulin levels in HFD-fed mice. Finally, we show that CE and PE treatment restored to normal the hyperlipidemia- and hyperglycemia-related gene expressions in HFD-fed mice. These results indicate that dietary CE or PE exert their anti-obesity and antidiabetic effects by regulating adipogenesis and insulin signaling pathways. Finally, we show that dietary CE or PE have similar anti-obesity and antidiabetic effects even when included in a normal mouse diet. Although cordycepin is not found in PE, PE treatment improves lipid and glucose metabolic parameters in a manner similar to CE. We find that PE provides alternative potential therapeutic treatments for obesity and diabetes. [ABSTRACT FROM AUTHOR]

• Antioxidant capacity of Apis mellifera major royal jelly proteins (AmMRJPs) was assayed. • AmMRJPs reduce caspase-3 activity and oxidative stress-induced cell apoptosis. • AmMRJPs exhibit DPPH radical-scavenging activity. • mMRJPs protect against oxidative DNA damage. Major royal jelly proteins (MRJPs) of honeybee royal jelly (RJ) exhibit antimicrobial and antioxidant activities. Although MRJPs of Apis mellifera RJ (AmMRJPs) responsible for antibacterial activity have been identified, AmMRJPs with antioxidant effects remain to be elucidated. Here we identified and compared the antioxidant activities of purified recombinant AmMRJPs 1–7, which are expressed in baculovirus-infected insect cells. Antioxidant assays of recombinant AmMRJPs 1–7 against H 2 O 2 revealed that AmMRJPs reduce caspase-3 activity and oxidative stress-induced cell apoptosis and lead to increased cell viability. Consistent with these results, AmMRJPs 1–7 exhibit 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and protect against oxidative DNA damage. These results indicate that AmMRJPs play a role as antioxidants in A. mellifera RJ. [ABSTRACT FROM AUTHOR]

The giant mealworm Zophobas atratus , which is used for commercial purposes, was officially introduced to Korea. However, physiological information of oocyte maturation for the mass rearing of Z. atratus has not been investigated. Here, we cloned Z. atratus vitellogenin (ZaVg) cDNA and analyzed the expression profiles during development to better understand oocyte maturation in Z. atratus. A multi-alignment and phylogenetic analysis using the deduced amino acids of ZaVg and those of other insect vitellogenins showed that ZaVg has typical Coleopteran Vg features. Consistent with ovarian development, lower expression levels of ZaVg protein in the fat body, hemolymph, and ovary were observed at 0 and 10 days in the adult stage, while higher expression levels of that were detected at 20, 30, and 40 days in the adult stage. These results suggest that the expression level of ZaVg is adult-stage specific, which providing basic information on Vg and oocyte development in Z. atratus. Unlabelled Image • The cDNA of the giant mealworm (Zophobas atratus) vitellogenin (ZaVg) was cloned. • The structural and phylogenetic analyses of insect vitellogenins were compared. • ZaVg was observed in the fat body, hemolymph, and ovary after adult eclosion. • ZaVg is related to ovarian maturation. [ABSTRACT FROM AUTHOR]

Major royal jelly proteins (MRJPs) are the protein components in royal jelly (RJ). MRJPs 1–7 are detected in the honeybee Apis mellifera RJ. Although A. mellifera MRJP (AmMRJP) 2 exhibited antibacterial activity, the other MRJPs with antimicrobial activities in A. mellifera RJ remains largely unknown. Here, we compared the antibacterial activity of recombinant AmMRJPs 1–7 expressed in baculovirus-infected insect cells. Antibacterial assays of recombinant AmMRJPs 1–7 against the gram-negative bacterium Escherichia coli revealed that AmMRJPs 2–5 and 7 exhibited antibacterial activity, whereas AmMRJPs 1 and 6 displayed almost no antibacterial activity. Consistent with the antibacterial activity of AmMRJPs, AmMRJPs 2–5 and 7 are bound to bacterial cell walls. These results indicated that AmMRJPs 2–5 and 7 contribute directly to the antibacterial property of RJ, suggesting that MRJPs play a role in the antimicrobial property of RJ. Unlabelled Image • Recombinant Apis mellifera major royal jelly proteins 1–7 (AmMRJPs 1–7) were produced. • Antibacterial activity of recombinant AmMRJPs 1–7 was compared. • AmMRJPs 2–5 and 7 exhibit antibacterial activities against Escherichia coli. • AmMRJPs 2–5 and 7 directly contribute to the antibacterial property of RJ. [ABSTRACT FROM AUTHOR]

Royal jelly (RJ) is a well-known functional and medicinal food for human health promotion. Major royal jelly proteins (MRJPs), which are the major protein components in RJ, exhibit antimicrobial activities. However, the identities of the MRJPs of RJ responsible for its antioxidant effects have remained unclear. Here, we report that honeybee (Apis cerana) MRJP 2 (AcMRJP2) acts as an antimicrobial and antioxidant agent in RJ. Using recombinant AcMRJP2, which was produced in baculovirus-infected insect cells, we established the antimicrobial and antioxidant roles of MRJP 2. AcMRJP2 bound to the surfaces of bacteria, fungi, and yeast, which then induced structural damage in the microbial cell walls and led to a broad spectrum of antimicrobial activities. AcMRJP2 protected mammalian and insect cells via the direct shielding of the cell against oxidative stress, which led to reduced levels of caspase-3 activity and oxidative stress-induced cell apoptosis, followed by increased cell viability. Moreover, AcMRJP2 exhibited DNA protection activity against reactive oxygen species (ROS). Our data indicate that AcMRJP2 could play a crucial role as an antimicrobial agent and antioxidant in RJ, suggesting that MRJP 2 is a component responsible for the antimicrobial and antioxidant activities of RJ. Unlabelled Image • Apis cerana major royal jelly protein 2 (AcMRJP2) exhibits antimicrobial activities. • AcMRJP2 protects insect and mammalian cells against oxidative stress. • AcMRJP2 exhibits DNA protection activity against reactive oxygen species (ROS). • AcMRJP2 acts as an antimicrobial agent and antioxidant in royal jelly. [ABSTRACT FROM AUTHOR]

The protein component of honeybee royal jelly (RJ) is constituted by major royal jelly proteins (MRJPs). The Asiatic honeybee (Apis cerana) MRJP-4 (AcMRJP4) exhibits antimicrobial activities. In this study, we identified the antimicrobial activity of AcMRJP4-15, which is a hydrophilic peptide with 88 amino acid residues in the C-terminal of AcMRJP4 that contains a high content of Asn and positively charged amino acids. Recombinant AcMRJP4-15, which is expressed as a 15-kDa peptide in baculovirus-infected insect cells, induced structural damage to the cell walls of bacteria, fungi, and yeast. Interestingly, the antimicrobial activity of AcMRJP4-15 was greater than that of AcMRJP4, demonstrating that the antimicrobial activity of AcMRJP4 was due in large part to the C-terminal. Our data suggest that AcMRJP4-15 can function as an effective antimicrobial agent. Unlabelled Image • The C-terminal of Apis cerana major royal jelly protein 4 (AcMRJP4-15) was identified to have antimicrobial activity. • Antimicrobial activity of AcMRJP4-15 was significantly higher than that of AcMRJP4. • Antimicrobial activity of AcMRJP4 was mostly attributed to the C-terminal. • AcMRJP4-15 can function as an effective antimicrobial agent. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Unlabelled Image Highlights • The cDNA of the honeybee (Apis cerana) major royal jelly protein 4 (AcMRJP4) was cloned. • AcMRJP4 exhibits antimicrobial activities against bacteria, fungi, and yeast. • AcMRJP4 functions as an antimicrobial agent. [ABSTRACT FROM AUTHOR]

Abstract Centipede venom contains a variety of proteins, peptides, and enzymes. However, the biological actions of toxin peptides in centipede venom remain largely unknown. In this study, we identified a centipede (Scolopendra subspinipes mutilans) venom toxin peptide (SsmTP) that was shown to act as a cell growth factor at low concentrations-in vitro. SsmTP was found to consist of 66 amino acids that display seven cysteine residues, which exhibited high similarity to the predicted neurotoxin. SsmTP was expressed in the venom gland of S. s. mutilans. A recombinant SsmTP peptide of approximately 5.2 kDa was produced in baculovirus-infected insect cells. Interestingly, SsmTP exhibited cytotoxicity in murine cells in a concentration-dependent manner but displayed a cell growth effect at low concentrations. SsmTP at a low concentration also protected murine cells against oxidative damage through the inhibition of caspase-1 and apoptosis. Our data indicate that SsmTP acts as a cell growth factor and a toxin peptide in a concentration-dependent manner. Consequently, our results provide evidence that the identification of SsmTP, a centipede toxin peptide, may not only elucidate the biological action of toxin peptides but also offer additional insight into the pharmacological applications of centipede venoms. Graphical abstract Unlabelled Image Highlights • A centipede (Scolopendra subspinipes mutilans) venom toxin peptide (SsmTP) was identified. • SsmTP induces cytotoxicity at high concentrations. • SsmTP has a cell growth effect at low concentrations. • SsmTP acts as a cell growth factor and a toxin peptide in a concentration-dependent manner. [ABSTRACT FROM AUTHOR]

Insect immulectins are involved in various aspects of the innate immunity, including encapsulation, melanization, and phagocytosis. Although the silkworm Bombyx mori immulectin (BmIML) has been reported previously, the ligand and functions of BmIML have not been investigated. Here, we show the dual roles of BmIML in cuticular melanization and immunity of B. mori . BmIML recognizes carbohydrates in a Ca 2 + -dependent manner and binds to Gram-negative and Gram-positive bacteria, fungi, and yeast. BmIML was expressed in the fat body during infections and localized to the hemocytes of silkworms. Additionally, BmIML was expressed in the epidermis during the prepupal stage and localized to the cuticle of silkworms. After treatment with E. coli , dopa, dopamine, or tyrosine injections, BmIML production was induced in the fat body but not in the epidermis of silkworms. Treatment with BmIML RNAi resulted in the arrest of pupal cuticular melanization. Therefore, we concluded that BmIML is involved in pupal cuticular melanization and innate immunity responses of silkworms, suggesting dual roles for BmIML. [ABSTRACT FROM AUTHOR]

Our previous study demonstrated that the secapin peptide from the venom of the Asiatic honeybee ( Apis cerana , AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. In the present study, we investigated the anti-microbial activity and cytotoxicity of a synthetic AcSecapin-1 peptide. Seven synthetic AcSecapin-1 peptides (AcSecapin-S1 to AcSecapin-S7) were synthesized based on the peptide sequence of AcSecapin-1. AcSecapin-S1, which consists of the 25-amino acid sequence identical to that of the mature AcSecapin-1 peptide, exhibited the highest anti-microbial activity against bacteria and fungi. This was followed by AcSecapin-S6, which was missing 10 N-terminal amino acids and 6 C-terminal amino acids from AcSecapin-S1. Furthermore, AcSecapin-S1 was not cytotoxic and did not activate macrophages. Taken together, our data demonstrated that a synthetic AcSecapin-1 peptide could exhibit anti-microbial activity without producing a cytotoxic or inflammatory response, suggesting that this synthetic AcSecapin-1 peptide can be used as an anti-microbial agent for biomedical applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • Ingestion of peppermint oil (PO) upregulated CCHamide-2 in nurse bees. • Ingestion of heat-killed pathogens supplemented with PO induced vitellogenin and defensin-1 in nurse bees. • PO promoted the induction of vitellogenin and defensin-1 in nurse bees upon ingestion of heat-killed pathogens. Transgenerational immune priming (TGIP) in honeybees (Apis mellifera) has been attracting increasing attention as an efficient method to protect colonies from infectious diseases. This study investigated whether feeding nurse bees peppermint oil (PO) along with a cocktail of heat-killed Ascosphaera apis and Paenibacillus larvae (A + P + PO cocktail) can promote the induction of marker genes for TGIP. In the A + P + PO cocktail-fed nurse bees, CCHamide-2 was significantly upregulated in the brain, and the body weight was highly increased. Consequently, vitellogenin (Vg) and defensin-1 showed a significant upregulation in the fat body of nurse bees with the A + P + PO cocktail. Thus, our findings indicate that PO promoted the induction of Vg and defensin-1 in nurse bees upon ingestion of the A + P + PO cocktail. [ABSTRACT FROM AUTHOR]

Silk fibroin proteins serve as biomaterials for diverse applications. Previous studies have shown that silk fibroin proteins can be used in biomedical applications for treating diabetes. In the present study, we investigated the cytotoxicity and inflammatory response induced by spider silk fibroin protein in vitro. By using recombinant AvMaSp-R spider silk fibroin protein, which consists of the 240 amino acid repetitive domain of the spider ( Araneus ventricosus ) silk fibroin protein and was expressed in baculovirus-infected insect cells, we tested the cytotoxicity, apoptosis, macrophage stimulation, and release of proinflammatory mediators and cytokines in vitro. We found that recombinant AvMaSp-R was not cytotoxic and did not activate macrophages. Consequently, our results provide evidence that recombinant AvMaSp-R produces a non-cytotoxic and non-inflammatory response, suggesting that recombinant AvMaSp-R silk fibroin protein could be a biomaterial for biomedical applications. [ABSTRACT FROM AUTHOR]

Chitin, a structural component of the insect peritrophic membrane (PM), is associated with chitin-binding proteins (CBPs). Insect CBPs have been studied extensively regarding their interactions with chitin in the structure and functions of the cuticle and PM. However, the functions of bee CBPs are poorly understood. In this study, we present the molecular cloning and functional characterization of a CBP from the Asiatic honeybee Apis cerana . A. cerana CBP (AcCBP) contains the peritrophin-A domain with a six-cysteine motif. AcCBP is exclusively expressed in the midgut and is localized in the PM of A. cerana worker bees. Recombinant AcCBP was expressed in baculovirus-infected insect cells, and it exhibited chitin-binding ability, thus indicating a role for AcCBP as a CBP in the PM of A. cerana worker bees. Furthermore, AcCBP bound to entomopathogenic fungal surfaces but did not show anti-fungal activity. Taken together, our data demonstrate that AcCBP functions as a CBP of the bee PM. [ABSTRACT FROM AUTHOR]

Bee venom contains a variety of toxic components, including enzymes, peptides, and biogenic amines. An acid phosphatase Acph-1-like protein has been identified from Asiatic honeybee ( Apis cerana ) venom. However, no molecular information is currently available for acid phosphatases from A. cerana venom. In this study, an A. cerana venom acid phosphatase ( Ac VAP) was identified. The amino acid sequence analysis of the predicted Ac VAP protein revealed high identity with other bee venom acid phosphatases. An anti- Ac VAP antibody was produced against a recombinant Ac VAP (46-kDa) expressed in in baculovirus-infected insect cells. Northern and Western blot analyses showed that Ac VAP was expressed in the venom gland and was present as a 46-kDa protein in the secreted bee venom. The enzymatic properties of recombinant Ac VAP were determined using p -nitrophenyl phosphate ( p -NPP) as a substrate and exhibited the highest activity at pH 4.8 and 45 °C. Taken together, our data demonstrated that Ac VAP functions as a bee venom acid phosphatase. [ABSTRACT FROM AUTHOR]

The honeybee inhibitor cysteine knot (ICK) peptide acts as an antifungal peptide and insecticidal venom toxin. However, the ICK peptide from bumblebees has not been characterized. Here, we report the molecular cloning and antifungal activity of a bumblebee ( Bombus ignitus ) ICK peptide (BiICK). We identified a BiICK that contains an ICK fold. The BiICK was expressed in the epidermis, fat body, and venom gland of B. ignitus worker bees. A 6.7-kDa recombinant BiICK peptide was expressed in baculovirus-infected insect cells. Recombinant BiICK peptides directly bound to Beauveria bassiana , Ascosphaera apis , and Fusarium graminearum , but they did not bind to Escherichia coli , Paenibacillus larvae , or Bacillus thuringiensis . Consistent with this finding, BiICK exhibited antifungal activity against fungi. These results demonstrate that BiICK acts as an antifungal peptide. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Insect cuticular melanization is a defense mechanism involving the prophenoloxidase (proPO)-activating system. Here, we showed the differential regulation of tyrosine hydroxylase in silkworm ( Bombyx mori ) pupae. B. mori tyrosine hydroxylase (BmTH) was expressed in the epidermis during development or in the fat body during infection, which indicates that the differential expression of BmTH is responsible for cuticular melanization or innate immunity. The melanin of the silkworm pupal cuticle formed a melanization complex with Beauveria bassiana and this effect showed an antifungal activity, indicating that melanin in silkworm pupal cuticle plays a role in the defense against fungal pathogens. Finally, we found that the differential expression of BmTH is involved in pupal cuticular melanization and adult pigmentation as well as the innate immunity of silkworms, demonstrating a differential regulation of BmTH in a tissue-specific manner. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors play a critical role in physiological processes and immune responses by regulating serine protease activities. Here we describe the molecular cloning and antimicrobial activities of a serine protease inhibitor from the hornfaced bee, Osmia cornifrons (OcSPI). OcSPI consists of 405 amino acid residues and contains a potential reactive center loop (RCL) region in its C-terminus. Recombinant OcSPI was produced as a 64-kDa glycoprotein in baculovirus-infected insect cells and exhibited inhibitory activity against chymotrypsin. Additionally, OcSPI demonstrated inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, but not against tissue plasminogen activator, thrombin, or plasmin. Recombinant OcSPI bound directly to Escherichia coli , Bacillus subtilis , and Beauveria bassiana and exhibited antimicrobial activity against both bacteria and fungi. Our results demonstrated the antimicrobial functions of OcSPI and suggest a role for OcSPI in the immune response of O. cornifrons bees. [ABSTRACT FROM AUTHOR]

Cystatins are involved in various physiological and cellular processes, including immune responses, protein homeostasis, signaling pathways, and apoptosis. Thus far, no Cystatins have been identified from spider venom. In this study, we report the cloning and characterization of a spider venom-derived Cystatin from Araneus ventricosus (AvCystatin). The AvCystatin gene contains an open reading frame of 405 bp encoding a predicted protein of 134-amino acids with a 16-amino acid signal peptide. Sequence alignment and structural modeling indicated that AvCystatin is closely related to family 2 Cystatins. Endogenous AvCystatin was present as an 18-kDa peptide in spider venom. Recombinant AvCystatin, expressed in baculovirus-infected insect cells, showed inhibitory activity against papain (K i 86.73 nM), but not trypsin and chymotrypsin, defining a role for AvCystatin as a spider venom-derived cysteine protease inhibitor. Furthermore, recombinant AvCystatin exhibited stability against high temperatures and pH extremes, but had no effects on the growth of the entomopathogenic fungi Beauveria bassiana . These data demonstrate that AvCystatin is a novel member of the family 2 Cystatins and provide new insight for the future investigations of spider venom-derived Cystatins. [ABSTRACT FROM AUTHOR]

Novel combinations aiming at maximizing the efficacy of bortezomib are highly valued in the clinic. Therefore the current study investigated the outcomes of combining bortezomib with dipyridamole, a well-known antiplatelet. The co-treatment exerted a synergistic lethality in a panel of human leukemia/lymphoma cell lines of different origin. Mechanistically, dipyridamole did not modulate the proteasome inhibitory activity of bortezomib. However, dipyridamole triggered an endoplasmic reticulum (ER) stress, and co-treatment with bortezomib resulted in higher levels of ER stress than either monotherapies. Relieving ER stress with the protein translation inhibitor, cycloheximide suppressed cell death. Moreover, the enhanced ER stress by the co-treatment was associated with an aggravation of reactive oxygen species (ROS) generation and glutathione (GSH) depletion. Replenishing GSH pools significantly scavenged ROS and rescued the cells. Importantly, the cytotoxicity of the co-treatment was executed mainly via the mitochondrial apoptotic pathway with an efficient suppression of the key anti-apoptotic regulators, Mcl-1, Bcl-xl, Bcl-2 and XIAP, driving the independence of the co-treatment-induced apoptosis of a single apoptotic trigger. Furthermore, the intrinsic potential of bortezomib to inhibit important pro-survival pathways was enhanced by dipyridamole in a GSH/ROS-dependent manner. Interestingly, dipyridamole abrogated JAK2 phosphorylation indirectly and selectively in cancer cells, and the co-treatment-induced cytotoxicity was preserved in a model of stromal-mediated chemoresistance. In nude mice, the antitumor activity of the co-treatment surpassed that of bortezomib monotherapy despite that synergy was lacking. In summary, findings of the present study provided a preclinical rationale which warrants further clinical evaluation of bortezomib/dipyridamole novel combination in hematologic malignancies. [ABSTRACT FROM AUTHOR]

[Display omitted] • Apis mellifera has major royal jelly protein 8 and 9 (AmMRJP 8 and 9) in its venom. • AmMRJP 8 and 9 inhibit microbial serine proteases. • AmMRJP 8 and 9 exhibit antibacterial and antifungal activities. • AmMRJP 8 and 9 act as antimicrobial agents in honeybee venom. Honeybee venom is a complex mixture of toxic components, including major royal jelly protein (MRJP) 8 and 9. MRJP 8 and MRJP 9 are allergens, and MRJP 8 reduces melittin-induced cell apoptosis. However, their functional roles are poorly understood, and their antimicrobial activities have not been determined. In this study, the antimicrobial role of MRJP 8 and MRJP 9 of honeybee (Apis mellifera) venom (AmMRJP 8 and AmMRJP 9) was demonstrated. The presence of AmMRJP 8 and AmMRJP 9 in the secreted venom was observed using antibodies against recombinant AmMRJP 8 and AmMRJP 9 produced in baculovirus-infected insect cells. Recombinant AmMRJP 8 and AmMRJP 9 exhibited an inhibitory activity against microbial serine proteases. Consistent with their inhibitory activity, they induced structural damage by binding to microbial surfaces, resulting in a broad-spectrum antimicrobial activity against bacteria and fungi. They had little effect on hemolysis. Therefore, AmMRJP 8 and AmMRJP 9 could function as antimicrobial agents in honeybee venom. [ABSTRACT FROM AUTHOR]

Abstract: Spider silks have great potential as biomaterials with extraordinary properties. Here, we report the cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA encoding the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. An analysis of the cDNA sequence shows that AvMaSp consists of a 240 amino acid repetitive region and a 99 amino acid C-terminal non-repetitive domain. The peptide motifs that were found in the spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin family of proteins. The AvMaSp-R cDNA, which encodes the 240 amino acid repetitive domain, was expressed as a soluble 22kDa polypeptide in baculovirus-infected insect cells. Recombinant AvMaSp-R was degraded abruptly by trypsin. However, AvMaSp-R was stable at 100°C for at least 30min. Additionally, the AvMaSp-R was stable at pH values from 2 to 12 for at least 1h. Taken together, our findings describe the molecular structure and biochemical properties of the A. ventricosus major ampullate silk protein and demonstrate its potential as a biomaterial. [Copyright &y& Elsevier]

Abstract: The expression of viral antigens in baculovirus-infected insect cells is often ineffective. As an alternative approach, therefore, we developed the recombinant polyhedra technology, which is an efficient strategy for the production of viral subunit vaccine. Here, we report a strategy for the large-scale production of a pseudorabies virus (PRV) gB or gC in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the epitope regions of PRV gB or heparin-binding domains of PRV gC. Recombinant BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae expressed native polyhedrin and fusion protein that was detected using both anti-polyhedrin and anti-PRV gB or anti-PRV-gC antibodies. Electron and confocal microscopy demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin with a normal morphology and that the recombinant polyhedra contained PRV gB or gC. The yield of gB or gC antigen produced in BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae reached 0.69 or 0.46mg per larva, respectively, at 6days post-infection. These results demonstrate that the recombinant polyhedra strategy can be used for the large-scale production of PRV gB or gC antigen. [Copyright &y& Elsevier]

Abstract: Cathepsin D is involved in the metamorphosis of the silkworm, Bombyx mori. Here, we show the expression profile of B. mori cathepsin D (BmCatD) in the fat body during exposure to stressors, such as high temperature and H2O2. Exposure of larvae in the fifth instar stage to high temperature (28°C) led to accelerated metamorphosis and shortened larval stage compared to control larvae grown at 23°C. Concomitantly, the expression level of BmCatD mRNA was greatly increased during exposure to high temperature. We also detected significantly elevated H2O2 levels in the hemolymph of larvae treated with high temperature. To confirm that oxidative stress induces BmCatD expression, B. mori larvae were injected with H2O2. As predicted, we observed increased expression of BmCatD following H2O2 exposure. Based on these results, we conclude that BmCatD expression is induced by high temperature and H2O2 exposure and that this stress-induced BmCatD expression leads to early metamorphosis. [Copyright &y& Elsevier]