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Mitohormesis defines the increase in fitness induced by adaptive responses to mild mitochondrial stress. Here, we show that N-acetyloxfenicine (NAO) exerted higher thermotolerance than an endogenous mitohormesis-inducer, N-acetyltyrosine. This activity was not observed in armyworm larvae injected with oxfenicine, suggesting the importance of N-acetylation. NAO-induced hormetic effect was triggered by transient perturbation of mitochondria, which causes a small increase in ROS production and leads to retrograde responses including enhanced expression of antioxidant enzyme genes via activation of FoxO transcription factors. Furthermore, pretreatment with NAO significantly repressed stress-induced peroxidation of lipids in mice and growth of colorectal cancer HCT116 cells that had been transplanted into nude mice. Taken together, NAO is a potent mitohormesis-inducer that is similar to N-acetyltyrosine in terms of structure and functions.

A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. We further report that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these various homeostatic attributes of GBP: We found that elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan. We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity.

Insect cytokine growth blocking peptide (GBP) is synthesized as an inactive precursor, termed proGBP, that is normally present in a significant concentration in the hemolymph of non-stressed animals (Hayakawa, 1990, 1991). Under stress conditions, proGBP is instantly processed to active GBP by a serine protease and this is thought to be an important initial step for insects to cope with stress-induced adverse effects via GBP-induced physiological changes. However, the detailed mechanism underlying proteolytic processing of hemolymph proGBP in insects under stress conditions remains unknown. Here we demonstrated that proGBP processing requires ROS-induced release of a proteinaceous factor from hemocytes that activates the inactive proGBP processing enzyme. The release of the activator protein from hemocytes is initiated by an elevation of the cytoplasmic Ca2+ concentration induced by ROS. Therefore, we concluded that stress-induced activation of proGBP requires ROS-dependent stimulation of an intracellular calcium signaling pathway in hemocytes, followed by release of the hemocyte proteinaceous factor that specifically activates the proGBP processing enzyme.

Background Facultative parthenogenesis, seen in many animal phyla, is a reproductive strategy in which females are able to generate offspring when mating partners are unavailable. In some subsocial and eusocial insects, parthenogenesis is often more prevalent than sexual reproduction. However, little is known about how social cooperation is linked to the promotion of parthenogenesis. The domiciliary cockroach Periplaneta americana is well-suited to addressing this issue as this species belongs to the superfamily Blattoidea, which diverged into eusocial termites and shows facultative parthenogenesis. Results We studied environmental factors that influence asexual production of ootheca using behavioral assays in P. americana. When more than three virgin females immediately after the imaginal molt were kept together in a small sealed container, they tended to produce egg cases (oothecae) via parthenogenesis earlier than did isolated females, resulting in apparent synchronization of ootheca production, even among females housed in different containers. In contrast, virgin females housed with genitalia-ablated males or group-housed females with antennae ablated did not significantly promote ootheca production compared to isolated females. Daily addition of the primary sex pheromone component to the container did not promote ootheca production in isolated females. Another line of study showed that grouped females make parthenogenesis more sustainable than previously known; a founder colony of 15 virgin females was sufficient to produce female progeny for a period of more than three years. Conclusions Group-housed females promote and stabilize asexual ootheca production compared to isolated females, and that this promotion is triggered by female-specific chemosensory signals (other than sex pheromone) primarily detected by antennae. Promotion of ootheca production between females is likely to be an early stage of social cooperation, reminiscent of the foundation and maintenance of a colony by female pairs in the eusocial termite Reticulitermes speratus. Electronic supplementary material The online version of this article (doi:10.1186/s40851-017-0063-x) contains supplementary material, which is available to authorized users.

Invertebrates are productive models for understanding basic molecular principles that link cytokine-mediated inflammatory pathways to aging in humans. Here, we use a high-throughput dsRNA screen to determine that the multifunctional Drosophila cytokine, GBP, is a ligand for the hitherto orphan GPCR, Mthl10. Through genetic manipulation of the GBP/Mthl10 axis in larvae and adult flies, we are able to demonstrate how organismal longevity is interconnected to immunological, metabolic, and stress-protective responses. Our results provide a molecular basis for hypothesizing that a successful defense against environmental insults—be they pathogenic inflammation or noninfective challenges—will ultimately reduce lifespan.

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.

Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)-catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2-4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti-PPO antibody. We were unable to detect PPO-mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO-mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders.

Growth rates of immature animals are governed by their feeding activities. A reduction in feeding sometimes causes serious growth retardation in insects; a typical case is often seen in host insects parasitized by a solitary endoparasitoid wasp. However, understanding of the mechanisms underlying the physiological repression of parasitized insects is fragmentary. Here we analyzed brain gene expression of the host common cutworm, Spodoptera litura, parasitized by a solitary endoparasitoid, Microplitis manilae, and identified a novel gene whose expression was significantly enhanced by parasitization. The gene encoded a pre-pro-peptide of a cytokine-like molecule and its expression was observed mainly in nervous tissues, hemocytes, and integuments. The 25 amino acid cytokine-like peptide encoded by the C-terminus of this gene was demonstrated to exist in the hemolymph of S. litura larvae and to change hemocytes from non-adhesive to adhesive in vitro. Further, injection of the active peptide reduced feeding activities of test larvae and consequently delayed their growth. The enhanced gene expression was also observed in larvae under severe stress conditions: abdominal ligature, proleg cutting, mechanical vibration, low temperature, and heat shock at 45°C. Elevated gene expression was maintained only in seriously growth-retarded larvae but not in recovered larvae at 24h or 48h after heat treatment. Thus, it is reasonable to conclude that stress-induced elevation of the peptide gene expression highly correlates with reduced feeding activities and growth retardation of the host larvae parasitized by M. manilae. Based on the conclusion, we named this peptide stress-responsive peptide (SRP).

Recovery from weight loss after stress is important for all organisms, although the recovery mechanisms are not fully understood. We are working to clarify these mechanisms. Here, we recorded enhanced feeding activity of Drosophila melanogaster larvae from 2 to 4 h after heat stress at 35°C for 1 h. During the post-stress period, expression levels of sweet taste gustatory receptor genes (Grs), Gr5a, Gr43a, Gr64a, and Gr64f, were elevated, whereas bitter taste Grs, Gr66a, and Gr33a, were decreased in expression and expression of a non-typical taste receptor Gr, Gr68a, was unchanged. Similar upregulation of Gr5a and downregulation of Gr66a was recorded after cold stress at 4°C. Expression levels of tropomyosin and ATP synthase ß subunit were significantly increased in larval mouth parts around 3 to 5 h after the heat stress. We infer that up-regulation of post-stress larval feeding activity, and weight recovery, is mediated by increasing capacity for mouth part muscular movements and changes in taste sensing physiology. We propose that Drosophila larvae, and likely insects generally, express an efficient mechanism to recover from weight loss during post-stress periods.

The beta-1,3-glucan recognition protein (betaGRP)/Gram-negative bacteria-binding protein 3 (GNBP3) is a crucial pattern-recognition receptor that specifically binds beta-1,3-glucan, a component of fungal cell walls. It evokes innate immunity against fungi through activation of the prophenoloxidase (proPO) cascade and Toll pathway in invertebrates. The betaGRP consists of an N-terminal beta-1,3-glucan-recognition domain and a C-terminal glucanase-like domain, with the former reported to be responsible for the proPO cascade activation. This report shows the solution structure of the N-terminal beta-1,3-glucan recognition domain of silkworm betaGRP. Although the N-terminal domain of betaGRP has a beta-sandwich fold, often seen in carbohydrate-binding modules, both NMR titration experiments and mutational analysis showed that betaGRP has a binding mechanism which is distinct from those observed in previously reported carbohydarate-binding domains. Our results suggest that betaGRP is a beta-1,3-glucan-recognition protein that specifically recognizes a triple-helical structure of beta-1,3-glucan.

In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with well-characterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6h post challenge, and was somewhat elevated at 24h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis. By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum.

Desiccate (Desi), initially discovered as a gene expressing in the epidermis of Drosophila larvae for protection from desiccation stress, was recently found to be robustly expressed in the adult labellum; however, the function, as well as precise expression sites, was unknown. Here, we found that Desi is expressed in two different types of non-neuronal cells of the labellum, the epidermis and thecogen accessory cells. Labellar Desi expression was significantly elevated under arid conditions, accompanied by an increase in water ingestion by adults. Desi overexpression also promoted water ingestion. In contrast, a knockdown of Desi expression reduced feeding as well as water ingestion due to a drastic decrease in the gustatory sensillar sensitivity for all tested tastants. These results indicate that Desi helps protect insects from desiccation damage by not only preventing dehydration through the integument but also accelerating water ingestion via elevated taste sensitivities of the sensilla.

Using various types of DNAs prepared from plasmid DNA, complete double-stranded DNA (ds.DNA) with linear and cyclic forms and double-stranded DNA coexisting with single-stranded DNA (ss.DNA), the structure and fibrillogenesis of the collagen-DNA complex were investigated by means of turbidity, transmission electron microscopy, and confocal laser-scanning microscopy. The rate of fibrillogenesis of the collagen-DNA complex significantly depends on the DNA structure. The structure of the fibrils formed in the complexes showed a marked difference between the ds.DNA and ss.DNA complexes with collagen. Spatial distribution of the DNA and collagen in the complexes suggests that the characteristic collagen-DNA interaction depends on the DNA forms.

Several species and varieties of genus Cypripedium grown in Japanese Islands and northeast China were investigated using genetic analysis. C. macranthos var. hotei-atsmorianum and C. macranthos var. rebunense were collected in northern Japan. They are regarded as varieties or forms of C. macranthos of Eurasia. Although the large subunit of ribulose-1, 5-diphosphate decarboxylase/oxydase (rbcL) of chloroplast did not show any difference among the species, a difference between C. macranthos var. hotei-atsmorianum and the other two was found in the sequence of nuclear DNA encoding the internal transcribed spacer (ITS) region of ribosomal RNA. Random amplified polymorphic DNA method (RAPD) revealed a difference between C. macranthos var. rebunense from others. That C. calceolus and C. macranthos which were both collected in the Eurasia differed was demonstrated by their rbcL analyses and ITS sequences. The genetic analyses confirmed that C. × ventricosum was a hybrid of C. calceolus × C. macranthos. Thus, analyses using rbcL and ITS region are useful to distinguish species and varieties of Cypripedium.

Ca2+-calmodulin (CaM)-binding proteins in rat testes were characterized by assays for CaM-binding activity using the CaM-overlay method on transblots of electrophoresed gels and purification by gel-filtration, ion exchange, and adsorption chromatographies. A major CaM-binding protein complex (CaMBP) was identified and found to be comprised of three proteins with molecular masses 110, 100, and 70 kDa. Amino acid sequence analyses of lysylendopeptidase digests from these proteins indicated that all of the constituents of CaMBP are very similar to the members of the heat-shock protein family, i.e., the 110-kDa protein is similar to the APG-2/94 kDa rat ischemia-responsive protein, the 100-kDa protein is similar to the rat counterpart of the mouse APG-1/94 kDa osmotic stress protein, and the 70-kDa protein is similar to the rat testis-specific major heat-shock protein (HSP70). Immunohistochemistry using anti-CaMBP and anti-CaM antibodies demonstrated that CaMBP was co-localized with CaM in the cytoplasm of pachytene spermatocytes and nuclei of round spermatids. In addition, CaMBP, but not CaM, was localized at a high level in the residual bodies of elongated spermatids. The possible relevance of CaMBP to regulation of cell cycle progression and spermatogenesis is discussed in this paper. Mol. Reprod. Dev. 69: 316–324, 2004. © 2004 Wiley-Liss, Inc.

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.

Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)-catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2-4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti-PPO antibody. We were unable to detect PPO-mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO-mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders.

The β-1,3-glucan recognition protein (βGRP) has strong specific affinity for β-1,3-glucan, a component of the fungal cell wall. Its interaction with β-1,3-glucan initiates the activation of the prophenoloxidase cascade, which is an important defense system in invertebrates of many species. We cloned the cDNA of the βGRP of the silkworm Bombyx mori. The βGRP mRNA transcript was constitutively expressed in the hemocytes, fat body, and epithelial cells of the naive silkworm. At the same time, a bacterial or yeast challenge was indicated to intensify the transcription. Comparison of the deduced amino acid sequence with known sequences revealed that the βGRP contained a region (Thr264 to Pro386) displaying significant similarity to the catalytic regions of bacterial β-1,3-glucanases and much higher similarity to the glucanase-like regions of Gram-negative bacteria-binding proteins found in the silkworm B. moriand the mosquito Anopheles gambiae. The region (Thr264 to Pro386) of the βGRP, however, was demonstrated not to have appreciable affinity for β-1,3-glucan. A recombinant peptide corresponding to an N-terminal region (Tyr1 to Ala102) of the βGRP bound strongly to β-1,3-glucan. These results indicate that the binding domain of the βGRP for β-1,3-glucan is located in the N-terminal region. Glucanases and the current pattern-recognition proteins that contain a glucanase-like region seem to have a common origin in their molecular evolution.

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect. The PGRP is an essential component for peptidoglycan to trigger the prophenoloxidase cascade that is now recognized to be an important insect defense mechanism. We cloned cDNA encoding PGRP from the silkworm fat body cDNA library. Northern blot analysis showed that the PGRP gene is constitutively expressed in the fat body, epithelial cell, and hemocytes of naive silkworms. Furthermore, a bacterial challenge intensified the gene expression, with the maximal period being from 6 to 36 h after infection. The upstream sequence of the cloned PGRP gene was shown to contain putativecis-regulatory elements similar to the NF-κB-like element, interferon-response half-element, and GATA motif element, which have been found in the promoters of the acute phase protein genes of mammals and insects. A homology search revealed that the homologs of silkworm PGRP are present in mice, nematodes, and bacteriophages. This suggests that the recognition of peptidoglycan as foreign is effected in both vertebrates and invertebrates by PGRP homologs with an evolutionally common origin.

Cartilage is unique among tissues of mesenchymal origin in that it is resistant to vascular invasion due to an intrinsic angiogenic inhibitor. During endochondral bone formation, however, calcified cartilage formed in the center of the cartilaginous bone rudiment allows vascular invasion, which initiates the replacement of cartilage by bone. The transition of cartilage from the angioresistant to the angiogenic status thus plays a key role in bone formation. However, the molecular basis of this phenotypic transition of cartilage has been obscure. We report here purification of an endothelial cell growth inhibitor from a guanidine extract of bovine epiphyseal cartilage. The N-terminal amino acid sequence indicated that the inhibitor was identical to chondromodulin I (ChM-I), a cartilage-specific growth-modulating factor. Purified ChM-I inhibited DNA synthesis and proliferation of vascular endothelial cells as well as tube morphogenesis in vitro. Expression of ChM-I cDNA in COS7 cells indicated that mature ChM-I molecules were secreted from the cells after post-translational modifications and cleavage from the transmembrane precursor at the predicted processing signal. Recombinant ChM-I stimulated DNA synthesis and proteoglycan synthesis of cultured growth plate chondrocytes, but inhibited tube morphogenesis of endothelial cells. In situ hybridization and immunohistochemical studies indicated that ChM-I is specifically expressed in the avascular zone of cartilage in developing bone, but not present in calcifying cartilage. These results suggest a regulatory role of ChM-I in vascular invasion during endochondral bone formation.

Polydnaviruses (PDVs) are unique symbiotic viruses associated with parasitoid wasps: PDV particles are injected into lepidopteran hosts along with the wasp eggs and express genes that interfere with aspects of host physiology such as immune defenses and development. Recent comparative genomic studies of PDVs have significantly improved our understanding of their origin as well as the genome organization. However, the structural features of functional PDV particles remain ambiguous. To clear up the structure of Cotesia kariyai PDV (CkPDV) particles, we focused on immunoevasive protein (IEP), which is a mediator of immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes, because it has been demonstrated to be present on the surface of the virus particle. We discovered that IEP tends to polymerize and constitutes a previously unidentified thin surface layer covering CkPDV particles. This outermost surface layer looked fragile and was easily removed from CkPVD particles by mechanical stressors such as shaking, which prevented CkPDV from expressing the encoded genes in the host target tissues such as fat body or hemocytes. Furthermore, we detected IEP homologue gene expression in the wasp's venom reservoirs, implying IEP has another unknown biological function in the wasp or parasitized hosts. Taken together, the present results demonstrated that female C. kariyai wasps produce the fragile thin layer partly composed of IEP to cover the outer surfaces of CkPDV particles; otherwise, they cannot function as infectious agents in the wasp's host. The fact that IEP family proteins are expressed in both venom reservoirs and oviducts suggests an intimate relationship between both tissues in the development of the parasitism strategy of the wasp.

A protease zymogen present in the plasma fraction of the hemolymph of silkworm, Bombyx mori, was purified to homogeneity as judged by SDS/PAGE and IEF/PAGE. An activating system for the zymogen was also isolated from the plasma fraction and was shown to be triggered by zymosan (yeast cell wall polysaccharide containing beta-1,3-glucan) or peptidoglycan. Using this system, the purified zymogen was activated and the active enzyme was purified to homogeneity. The physiological function of the zymogen or its active form is not yet known, but the active form was shown to have narrower substrate specificity than trypsin. Among 33 peptide derivatives examined, Boc-Gln-Arg-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec (Boc = tert-butoxycarbonyl, NH-Mec = 4-methylcoumaryl-7-amide) were the best and the second best substrates, respectively. The purified zymogen was determined to be a 39-kDa protein consisting of a single polypeptide. The active form of the zymogen was labeled with [3H]diisopropylfluorophosphate and was completely inactivated by (p-amidinophenyl)methanesulfonyl fluoride. The molecular mass of the [3H]-labeled enzyme was determined to be 38 kDa in SDS/PAGE under reducing conditions. These results indicate that the 39-kDa protein purified in the present study is a zymogen of a serine-type protease and that the activation of the zymogen occurs by limited proteolysis.

Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. We demonstrated that growth-blocking peptide (GBP) is a potent cytokine that regulates stressor-induced AMP expression in insects. GBP overexpression in Drosophila elevated expression of AMPs. GBP-induced AMP expression did not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket were essential, indicating that GBP signaling in Drosophila did not use the orthodox Toll or Imd pathway but used the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors was apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious.

Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to one another and to foreign surfaces. Although extensive structure-activity studies have been performed on the GBP and its mutants in Lepidoptera Pseudaletia separata , the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified an adaptor protein (P77) with a molecular mass of 77 kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)–like domain in the cytoplasmic region of the C terminus. Although P77 showed no capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after GBP was added to a plasmatocyte suspension. Tyrosine phosphorylation of P77 also was observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. Tyrosine phosphorylation of the integrin β subunit also was detected in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin β subunit, but also abolished GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae after infection with Serratia marcescens , indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrate that GBP signaling in plasmatocytes requires the adaptor protein P77, and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.

The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.

In order to examine the role of αC domains, especially the terminal region of it, of fibrinogen Aα chain in the fibrin gel formation, we prepared a recombinant fibrinogen, Aα570 fibrinogen. Aα570 fibrinogen is the fibrinogen that is truncated at Aα570 and lacks 40 amino acids at the terminus of the αC domain. We examined the thrombin-catalyzed polymerization by transmission spectroscopy and confocal laser scanning microscopy (CLSM). We found that Aα570 fibrinogen exhibited a significantly delayed aggregation showing the importance of the terminal region of the αC domain in the polymerization process. Contrary to the fact that the addition of glucose to the mixture of fibrinogen and thrombin results in a substantial delay of the lateral aggregation of protofibrils for the native fibrinogen, delaying effect due to the addition of glucose disappeared thoroughly in the case of Aα570 fibrinogen. Turbidity measurements dependent upon the wavelength in the time course of gelation showed that mass per unit fiber length of Aα570 fibrinogen decreased significantly compared to the native fibrinogen, and the lateral aggregation of protofibrils was hindered significantly. Those results are consistent with the CLSM measurements that the bundles of protofibrils of Aα570 fibrinogen are thinner and denser with more branching than those of the native one. It was confirmed that C-terminal region of the αC domain plays an important role in the lateral aggregation and glucose interferes the interacting process between the αC domains.

SummaryThe Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Spätzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Spätzle processing during the immune response has remained elusive for a decade. Here, we report a protease, called Spätzle-processing enzyme (SPE), required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Spätzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes.

Hepatocyte growth factor activator (HGF activator) is a serine protease which converts single-chain HGF to the active two-chain form. HGF activator purified from human serum has a molecular mass of 34 kDa and consists of two chains held together by a disulfide bond. The nucleotide sequence of HGF activator cDNA shows that HGF activator is derived from the COOH-terminal region of a precursor of 655 amino acids by proteolytic cleavage of the bonds between Arg372 and Val373 and between Arg407 and Ile408 and that the precursor consists of multiple domains homologous to those observed in blood coagulation factor XII. In this study, we identified the precursor of HGF activator in human plasma using an enzyme-linked immunosorbent assay system. The precursor was purified from plasma by a five-step procedure. The purified precursor did not activate single-chain HGF. The precursor was efficiently cleaved in vitro by thrombin, at the bond between Arg407 and Ile408, in the presence of negatively charged substances. The cleaved precursor activated single-chain HGF. These findings led us to conclude that HGF activator is present in plasma as an inactive zymogen and that the zymogen is activated by the cleavage of the bond between Arg407 and Ile408 by thrombin. Characteristic structural domains in the NH2-terminal region of the zymogen may be involved in the binding of the zymogen to negatively charged substances, which stimulates the activation of the zymogen by thrombin.

A monospecific antibody against beta-1,3-glucan recognition protein (a 62 kDa protein) of the larval silkworm prophenoloxidase activating system was used to study the localization of the protein. Among tissues from 5th instar larvae, only hemocytes and plasma were shown to contain a 62 kDa polypeptide immunoreactive with the antibody. Ultra-thin sections of the hemocytes were stained by an indirect immunogold staining method. Labelling occurred in the granules and cytoplasm of granulocytes and in the spherules and cytoplasm of spherulocytes. It was most conspicuous in granules of granulocytes and uniformly labelled spherules of spherulocyte, whereas no labelling was evident in prohemocytes, plasmatocytes and oenocytoids. The results are discussed in relation to the mode of recognition of fungi as non-self in insect hemocoel.

Silkworm plasma was passed over a peptidoglycan-Sepharose 4B column or a CPB column [CPB, curdlan type polysaccharide (beta-1,3-glucan) bead] in the absence of divalent cation and the effluents from the columns were named plasma-PG and plasma-CPB, respectively. Prophenoloxidase activating system in plasma-PG was triggered by beta-1,3-glucan but not by peptidoglycan and the system in plasma-CPB was triggered by peptidoglycan but not by beta-1,3-glucan, suggesting that the peptidoglycan-Sepharose 4B column and the CPB column remove peptidoglycan-receptor and beta-1,3-glucan-receptor, respectively, from plasma. This result indicates that both receptors exist as separate entities in silkworm plasma. It is suggested that plasma-PG and plasma-CPB may be used as specific reagents to detect minute amounts of beta-1,3-glucan and peptidoglycan.

The plasma fraction (referred to as plasma-CPB) of silkworm hemolymph, from which a protein with affinity to beta-1,3-glucan was specifically removed according to Yoshida et al. (Yoshida, H., Ochiai, M., and Ashida, M. (1986), Biochem. Biophys. Res. Commun. 141, 1177-1184), was used to develop a method for quantitating the beta-1,3-glucan recognition protein of the prophenoloxidase activating system. In principle, a sample was judged to contain beta-1,3-glucan recognition protein when that sample could restore the ability of the system in plasma-CPB to be triggered by beta-1,3-glucan. Purification procedures for the recognition protein from silkworm hemolymph consisted of fractionation with ammonium sulfate, chromatography on DEAE-Toyopearl, Affi-Gel-heparin, and Mono Q and Superose 12 on the fast protein liquid chromatography system of Pharmacia LKB Biotechnology Inc. About 2.03 mg of beta-1,3-glucan recognition protein was obtained from 300 ml of hemolymph. The purified beta-1,3-glucan recognition protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. beta-1,3-Glucan recognition protein had a molecular mass of 62 kDa composed of a single polypeptide and an isoelectric point of pH 4.3. It bound to curdlan beads (composed of beta-1,3-glucan with average particle size of 80 micron) in the absence of divalent cation, whereas its binding to glucans with beta(1----4)- or alpha(1----6)-glycosidic linkages was not detected under the experimental conditions. Elution of the beta-1,3-glucan recognition protein bound to curdlan beads could be achieved under strongly denaturing conditions (after incubation of the beads with sodium dodecyl sulfate and beta-mercaptoethanol in boiling water for 5 min), but elution at room temperature was poor. Since beta-1,3-glucan recognition protein is the only protein in silkworm plasma with strong affinity to beta-1,3-glucan and endows the prophenoloxidase activating system in plasma-CPB with the ability to be triggered by beta-1,3-glucan, it was concluded that binding of the purified beta-1,3-glucan recognition protein with beta-1,3-glucan causes the triggering of the prophenol-oxidase activating system in silkworm plasma. However, the nature of the activity that is generated as the result of binding is not yet known. The purified beta-1,3-glucan recognition protein bound to beta-1,3-glucan did not hydrolyze appreciably any of the 26 commercially available peptidyl-7-amino-4-methylcoumarins, substrates for various proteases.

Silkworm (Bombyx mori) hemocytes were fixed immediately after collection. Thin sections of the hemocytes were stained by an indirect immunogold staining method using rabbit anti-prophenoloxidase/IgG as a primary antibody and colloidal gold coated with goat anti-rabbit/IgG as a secondary antibody. Electron micrographs of the sections revealed that only plasmatocytes and oenocytoids have prophenoloxidase both in cytoplasm and nucleus whereas granulocytes, spherulocytes and prohemocytes do not have appreciable amounts of the proenzyme. Cytoplasmic inclusions of oenocytoids also contain the proenzyme. A wide variety of concentrations of prophenoloxidase was observed among oenocytoids. Plasmatocytes appeared to have less prophenoloxidase than any oenocytoids. Once materials in the granules of granulocyte were discharged into the plasma and formed coagula, they cross-reacted with antiprophenoloxidase/IgG, suggesting that prophenoloxidase was trapped in the coagula by unknown mechanisms. This observation is discussed in relation to the dispute concerning the presence of prophenoloxidase or phenoloxidase in the granulocyte.