Self-defense System Research Articles

Antioxidant enzyme immunoreactivity in rat von Ebner gland after nickel treatment

Our study was designed to clarify the role of antioxidant enzymes in the rat von Ebner gland during acute nickel toxicity. After treatment with nickel acetate, we monitored ultrastructural alterations in acinar and ductal cells, immunohistochemical staining for glutathione peroxidase (GPx) and glutathione S-transferases (GST mu and GST pi), and immunoreactivity for malondialdehyde (MDA). Immunoreactivity for MDA was present only in the acinar cells, and it was enhanced at 3 h after Ni treatment. In contrast, immunoreactivities for GPx and GSTs did not change in acinar cells but significantly increased in ductal cells after Ni treatment. Cytoplasmic vacuoles increased in acinar cells at 3 h after Ni treatment, but they almost completely disappeared at 24 h. No morphological changes were observed in taste bud cells from Ni-treated rats. Because lipid peroxidation, as monitored by immunoreactivity for MDA, was only transiently increased in the acinar cells, the enhanced antioxidant enzyme immunoreactivity in ductal cells of the von Ebner gland plays a crucial role in the self-defense system against nickel toxicity in the rat oral cavity.

Comparative efficacies in vitro of antibacterial, fungicidal, antioxidant, and herbicidal activities of momilatones A and B

Momilactones A (MA) and B (MB) are phytoalexins derived from rice plant (Oryza sativa) and were considered to be a part of the mechanism of rice self-defense system. The present study was to evaluate the comparative efficacies in vitro of antibacterial, fungicidal, antioxidant, and herbicidal activities of MA and MB. In general, MB shows higher antifungal, antibacterial, and herbicidal action than MA, although its antioxidant property was less than MA. In herbicidal trial, the IC50 values of MB against germination, shoot and root elongation of barnyardgrass and monochoria were 40.9, 45.5, and 27.5, and 27.1, 17.3, and 0.9 µg, respectively. For MA, these values were 40.3, 35.6, and 55.1, and 43.9, 24.3, and 0.5 µg, respectively. For antifungal activity, momilactone B (IC50: 1.2, 123.9, and 53.4 µg) exerted significantly greater inhibition than MA (IC50: 78.1, 198.1, and 95.3 µg) against Botrytis cinerea, Fusarium solani, Colletrotrichum gloeosporioides, respectively, except for Fusarium oxysporum that both MA and MB showed no marked difference. In addition, MB exhibited significantly stronger antibacterial activity than MA against Pseudomonus ovalis, Bacillus cereus, and Bacillus pumilus, whereas the inhibitory activity of the two compounds was similar against Escherichia coli. Both MA and MB exerted rather weak antioxidant activity (EC50 was 783.9 and 790.7 µg, respectively), of which MA showed a slightly stronger antioxidant activity than MB. This study is the first to examine antifungal, antibacterial, and antioxidant activities of two phytoalexins, as well as their comparative efficacies against growth of the noxious weeds barnyardgrass and monochoria.

A GPIIb/IIIa bioreactor for specific treatment of immune thrombocytopenic purpura, an autoimmune disease. Preparation, in vitro characterization, and preliminary proof‐of‐concept animal studies

Immune thrombocytopenic purpura (ITP) is an autoimmune disease that affects thousands of Americans each year. The resulting thrombocytopenia, which develops from destruction of platelets (PLT) by anti-PLT autoantibodies (APAb), is often associated with hemorrhagic complications. Existing therapies are not effective and are associated with significant morbidity. Recently, a new treatment modality using plasmapheresis with a Protein-A column has shown some clinical promise. Yet, although this method would remove the pathogenic APAb, it would also deplete protective antibodies, thereby weakening the body's self-defense system. Because about 80% of patients with ITP develop APAb against the GPIIb/IIIa antigens on PLT, a novel approach of attaching a GPIIb/IIIa-linked bioreactor with an extracorporeal circuit is suggested herein to achieve highly effective/specific APAb removal and overcome shortcomings of plasmapheresis in treating ITP. A hollow fiber-based bioreactor device was fabricated, and GPIIb/IIIa antigens were immobilized onto the inner lumens of the hollow fibers by using the epichlorohydrin activation method. An optimized bioreactor containing a loading of 1.63 mg GPIIb/IIIa/g fibers and adsorption capacity of 1.9 mg 7E3/g fibers was developed. Preliminary proof-of-concept investigation using a 7E3-induced thrombocytopenic rat model (which mimicked clinical ITP) was carried out. A complete (100%) return of PLT counts to their initial levels was observed in rats within 6 h after the GPIIb/IIIa bioreactor treatment. In addition, a rapid restoration of WBC counts in the treated rats was also found. These preliminary findings shed light of promise of using the GPIIb/IIIa bioreactor approach in achieving highly improved ITP therapy.

Antimicrobial peptides from marine invertebrates.

Marine invertebrates lack an acquired, memory-type immunity based on T-lymphocyte subsets and clonally derived immunoglobulins (72). This differs from the vertebrate immune system, which is characterized by somatic gene rearrangement, clonal selection, and expansion and a discriminative ability that includes lymphocytes, among other factors, which impart specificity and memory (71). Marine invertebrates rely solely on innate immune mechanisms that include both humoral and cellular responses. Humoral immunity in marine invertebrates is characterized by antimicrobial agents present in the blood cells and plasma (92), along with reactions such as hemolymph coagulation or melanization (79, 85). Cellular immunity in marine invertebrates is based on cell defense reactions, including encapsulation, nodule formation, and phagocytosis (92). The cellular component of marine invertebrate immunity is mediated by hemocytes, motile cells that phagocytize microbes and secrete soluble antimicrobial and cytotoxic substances into the hemolymph (53). This differs from insects, especially Drosophila melanogaster, which rely largely on the challenge-induced synthesis of antimicrobial peptides by the fat body (30, 88) and use exclusion, via a tough exoskeleton, as their major antimicrobial defense. The circulating hemolymph in marine invertebrates contains biologically active substances such as complement, lectins, clotting factors, and antimicrobial peptides (57). All of these factors contribute to a self-defense system in marine invertebrates against invading microorganisms, which can number up to 106 bacteria/ml and 109 virus/ml of seawater (2). The survival of marine invertebrates in this environment suggests that their innate immune system is effective and robust (52). Antimicrobial peptides are a major component of the innate immune defense system in marine invertebrates. They are defined as molecules less than 10 kDa in mass which show antimicrobial properties (12) and provide an immediate and rapid response to invading microorganisms (8). The major classes of antimicrobial peptides include (i) α-helices, (ii) β-sheet and small proteins, (iii) peptides with thio-ether rings, (iv) peptides with an overrepresentation of one or two amino acids, (v) lipopeptides, and (vi) macrocyclic cystine knot peptides (24). There is evidence that antimicrobial peptides are widespread in invertebrates (15), especially in tissues such as the gut and respiratory organs in marine invertebrates, where exposure to pathogenic microorganisms is likely. In spite of variations in structure and size, the majority of antimicrobial peptides are amphiphilic, displaying both hydrophilic and hydrophobic surfaces. These peptides generally act by forming pores in microbial membranes or otherwise disrupting membrane integrity (82), which is facilitated by their amphiphilic structure. This mode of action is unlikely to lead to the development of resistance (9, 58), although it must be noted that this presumption is debatable (10). Recently, cationic antimicrobial peptides have been reported to be involved in many aspects of innate host defenses, associated with processes such as acute inflammation (25). The value of antimicrobial peptides in innate immunity lies in their ability to function without either high specificity or memory, and their small size makes them easy to synthesize (72). In addition, many antibacterial peptides show remarkable specificity for prokaryotes with low toxicity for eukaryotic cells (97). This is a characteristic that has favored their investigation and exploitation as potential new antibiotics (97). The recent appearance of a growing number of bacteria resistant to conventional antibiotics has become a serious medical problem. To overcome this resistance, the development of antibiotics with novel mechanisms of action is a pressing issue (48). Endogenous antimicrobial peptides are exciting candidates as new antibacterial agents due to their broad antimicrobial spectra, highly selective toxicities, and the difficulty for bacteria to develop resistance to these peptides (11, 26, 47). The ocean covers 71% of the surface of the earth and contains approximately half of the total global biodiversity, with estimates ranging between 3 and 500 × 106 different species (28). Marine macrofauna alone comprise 0.5 to 10 × 106 species (23). Therefore, the marine environment, especially marine invertebrates that rely solely on innate immune mechanisms for host defense, is a spectacular resource for the development of new antimicrobial compounds. This minireview will encompass what is known about gene-encoded antimicrobial peptides from marine invertebrates, covering the phyla Arthropoda, Chordata, and Mollusca (Table ​(Table11). TABLE 1. Antimicrobial peptides from marine invertebrates

Super Catalytic Antibody and Antigenase

By immunizing ground-state peptides or proteins, we can produce super catalytic antibodies possessing serine protease-like characteristics. The unique feature of super catalytic antibodies is their ability to decompose a target molecule that is being killed. The authors have succeeded in preparing super catalytic antibodies that destroy (i) the HIV-1 envelope protein gp41, (ii) chemokine receptor CCR5 peptide, and (iii) Helicobacter pylori urease, etc. Some of them can degrade antigens at high catalytic reaction rates. Regarding their Km and kcat, super catalytic antibodies show intermediary values between that of enzymes (high Km and kcat) and that of antibodies (low Km and kcat [=0]). The catalytic function of an antibody mostly resides in its light chain. From mouse Vκ germline analysis, it became clear that super catalytic antibodies are generated from some discrete germlines such as bb1, cr1, cs1, bl1, bj2 and bd2. In these Vκ germlines, at least one catalytic triad composed of three amino acid residues, namely, Asp1, Ser27a and His93, is encoded. Namely, the antibody light chains (super catalytic antibodies) generated from the germlines are inherently able to enzymatically decompose antigens. Thus, such antibody light chains can be referred to as antigenase (antigen-decomposing enzyme) and may have arisen during the evolution of antibodies to acquire a higher ability than that of enzymes for developing a sophisticated self-defense system for survival.

Enhanced immunocytochemical expression of antioxidant enzymes in rat submandibular gland after normobaric oxygenation

In order to clarify the role of antioxidant enzymes in the male rat submandibular gland against short-term normobaric oxygenation, we performed immunocytochemical staining of manganese-containing superoxide dismutase (Mn-SOD), copper- and zinc-containing SOD (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase, and glutathione S-transferases (GST alpha, GST mu, and GST pi) between days 1 and 7 after normobaric oxygenation. Ultrastructural alterations and immunoreactivities for malondialdehyde (MDA), a lipid peroxidation-related molecule, of the acinar and ductal cells after the oxygenation were also investigated. Immunoreactivity for MDA was exhibited in the acinar cells throughout the experiment. On the other hand, immunoreactivity for the SODs, CAT, and GSTs was not altered, when compared to that of controls, but was significantly elevated in the granular, striated, and excretory ductal cells. Since an increase of lipid peroxidation as indicated by enhanced immunoreactivity for MDA was detected in the acinar and intercalated ductal cells, the results indicate that the enhanced antioxidant enzymes in the granular, striated, and excretory ductal cells play a crucial role in the self-defense system of the male rat submandibular gland against normobaric oxygenation.

Nuclear factor-kappa B is required for tumor necrosis factor-alpha-induced manganese superoxide dismutase expression in human endometrial stromal cells.

We recently found that manganese superoxide dismutase (Mn-SOD) is up-regulated by TNF alpha at the transcription level in human endometrial stromal cells (ESC) and that TNF alpha-induced Mn-SOD expression is mediated by protein kinase C (PKC)-dependent phosphorylation. This study was undertaken to investigate whether nuclear factor-kappa B (NF-kappa B), a transcription factor, is involved in Mn-SOD induction by TNF alpha or PKC in human ESC. Electrophoretic mobility shift assay revealed that TNF alpha (1 ng/ml) and phorbol 12-myristate 13-acetate (TPA; 0.4 micro M), PKC activator, caused marked increases in nuclear NF-kappa B DNA binding activity. Secondly, ESC were incubated with MG132 (proteasome inhibitor) or SN50 (inhibitor of translocation of NF-kappa B into the nucleus) in the presence of TNF alpha or TPA. TNF alpha and TPA significantly increased Mn-SOD activities and Mn-SOD mRNA levels, and those effects were completely inhibited by MG132 and SN50. TNF alpha alone caused no effect on cell viability, but in the presence of MG132, TNF alpha significantly decreased cell viability. This inhibitory effect of MG132 was blocked by simultaneous addition of N-acetyl-L-cysteine, an antioxidant. In conclusion, the present study showed the involvement of NF-kappa B in Mn-SOD induction by TNF alpha or PKC in human ESC. This phenomenon could be a self-defense system of ESC against TNF alpha-mediated oxidative stress.

An HR-induced tobacco peroxidase gene is responsive to spermine, but not to salicylate, methyl jasmonate, and ethephon.

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.

Molecular cloning and characterization of a cDNA encoding a transferrin homolog from Bombyx mori.

We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyx mori. The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.

Antitumor effect of medium-chain triglyceride and its influence on the self-defense system of the body.

Medium-chain triglyceride (MCT), long-chain triglyceride (LCT), and their mixture were compared in reference to both cytotoxic effect against human tumor cells and influence on the immune system. MCT showed more potent cytotoxicity than LCT. Continuous contact with MCT also inhibited the cytotoxic effect of lymphokine-activated killer (LAK) cells much more strongly than LCT. However, there is a discrepancy between the concentration of MCT, or the mixture, that could suppress the growth of tumor cells and the concentration that inhibited the cytotoxicity of LAK cells. Moreover, no damage was observed in PBL or LAK cells or in their cytotoxicity when the cells were incubated with TG for 2 h a day. Thus, short-term contact with TG could inhibit tumor growth while immune system was maintained within normal range. Clinically fine control of the concentration of injected triglycerides, especially MCT, can be expected to provide potent antitumor effect and maintenance of normal immune system.

Hypercholesterolemia impairs a detoxification mechanism against peroxynitrite and renders the vascular tissue more susceptible to oxidative injury.

Previous studies have shown that glutathione (GSH) plays a central role in the protection against peroxynitrite (ONOO-) toxicity. The present study evaluated the changes of the GSH cytoprotective system against ONOO- in hypercholesterolemia and determined the effects of carvedilol, a beta-blocker with free radical-scavenging activity, on these hypercholesterol-induced changes. New Zealand White rabbits were fed either a normal diet, a high-cholesterol diet, or a high-cholesterol diet supplemented with either carvedilol or propranolol. Eight weeks later, the rabbits were killed, and the thoracic aortas were isolated. Total GSH content of aortic tissue, vasorelaxation response of aortic rings to exogenous ONOO-, No regeneration from ONOO- by aortic homogenate, and ONOO(-)-induced aortic tissue injury were examined. Hypercholesterolemia decreased tissue GSH content (0.52 +/- 0.08 versus 0.86 +/- 0.04 mumol/g in control, P < .01), attenuated the vasorelaxation response to ONOO- (40 +/- 4.1% versus 76 +/- 3.2%, P < .01), reduced NO regeneration from ONOO- (387 +/- 40 versus 662 +/- 51 pmol, P < .01), and potentiated ONOO(-)-induced vascular tissue injury (37 +/- 4.4% versus 14 +/- 2.6% of increase in lactate dehydrogenase release after 3-morpholinosydnonimine exposure, P < .01). Treatment of the hypercholesterolemic rabbits with carvedilol, but not propranolol, significantly preserved tissue GSH content (0.79 +/- 0.05 mumol/g, P < .01 versus nontreated hypercholesterolemic rabbits), restored the vasorelaxation to ONOO- (61 +/- 2%, P < .01), increased NO regeneration from ONOO- (583 +/- 39 pmol, P < .01), and attenuated ONOO(-)-induced tissue injury (19 +/- 1.8%, P < .01). These results suggest that hypercholesterolemia impairs the GSH-mediated detoxification mechanism against ONOO- and renders the vascular tissue more susceptible to oxidative injury. Carvedilol, a novel vasodilating beta-blocker with antioxidant activity, significantly preserved this self-defense system and protected tissue from oxidant injury.

Formation of adrenochrome from epinephrine by myeloperoxidase via a free radical: Its biological significance.

Adrenochrome, 2, 3-dihydro-3-hydroxy-1-methyl-1H-indole-5, 6-dione, is an unstable red compound formed by the oxidation of epinephrine, (R) -4- [1-hydroxy-2- (methylamino) ethyl] -1, 2-benzenediol. A red pigment was generated during the reaction between epinephrine and hydrogen peroxide by myeloperoxidase via a short-lived intermediate having a free radical detected by electron paramagnetic (spin) resonance spectroscopy, possibly o-semiquinone free radical of the substrate (epinephrine), in a moderately alkaline pH buffer solution. The light absorption peaks of the pigment at 220, 302 and 485 nm were identical with those of adrenochrome, and the Rf-value of the pigment on thin layer chromatography was consistent with that of standard adrenochrome. From these findings, the pigment was concluded to be adrenochrome. The biological significance of the formation of adrenochrome by the myeloperoxidase system in vivo might relate to a possible role in the body's self-defense system: myeloperoxidase released from neutrophils may catalyze the conversion of epinephrine to adrenochrome at a site of trauma or phlogosis, and this adrenochrome would promote wound healing through the formation of a blood clot and/or scab by radical polymerization of body-fluid components around the affected part, resulting in acceleration of hemostasis.

Passive range estimation using dual‐baseline triangulation

Modern combat systems based on active radar sensing suffer disadvantages against low-flying targets in cluttered backgrounds. Use of passive infrared sensors with these systems, either in cooperation or as an alternative, shows potential for improving target detection and declaration range for targets crossing the horizon. Realization of this potential requires fusion of target position data from dissimilar sensors, or passive sensor measurement of target range. The availability of passive sensors that can supply both range and bearing data on such targets would significantly extend the robustness of an integrated ship selfdefense system. We consider a new method of range determination with passive sensors based on the principle of triangulation, extending the principle to two orthogonal baselines. The performance of single or double baseline triangulation depends on sensor bearing precision and direction to target. An expression for maximum triangulation range at a required accuracy is derived as a function of polar angle relative to the center of the dual-baseline system. Limitations in the dual-baseline model due to the geometrically assessed horizon are also considered.

Nucleotide Sequence of 5′-Upstream Region and Expression of a Silkworm Gene Encoding a New Member of the Attacin Family

A genomic clone encoding a new member of attacin, an insect antibacterial protein, was isolated from a genomic library of the silkworm, Bombyx mori, and the nucleotide sequence of the 5′-upstream region was determined. The region contained Bm 1, a highly repetitive element of B. mori and a lipopolysaccharide (LPS) response element (RE)(NF-κB binding site), CAAT box and TATA box. Northern blot analysis showed that the attacin gene expression was rapidly induced by bacterial cell wall components such as LPS from Escherichia coli and peptidoglycan (PG) from Micrococcus luteus, suggesting that attacin plays an important role in an early phase of the self-defense system upon bacterial infection.

Biochemical composition of human saliva in relation to other mucosal fluids.

This paper describes several salivary components and their distribution in other mucosal secretions. Histatins are polypeptides which possess exceptional anti-fungal and anti-bacterial activities, but are nevertheless present only in saliva. Proline-rich proteins (PRPs) are members of a closely related family, of which the acidic PRPs are found solely in saliva, whereas the basic PRPs are also found in other secretions. Mucins are a group of glycoproteins that contribute to the visco-elastic character of the mucosal secretions. Despite the similarities in their structure and behavior, mucins have distinct tissue distributions and amino acid sequences. Other salivary proteins are present in one or more mucosal secretions. Lysozyme is an example of a component belonging to an ancient self-defense system, whereas secretory immunoglobulin A (sIgA) is the secreted part of a sophisticated adaptive immune system. Cystatins are closely related proteins which belong to a multigene family. Alpha-Amylase is a component that is believed to play a specific role in digestion, but is nevertheless present in several body fluids. Kallikrein and albumin are components of blood plasma. But whereas albumin diffuses into the different mucosal secretions, kallikrein is secreted specifically by the mucosal glands. The presence of these proteins specifically in saliva, or their distribution in other mucosal secretions as well, may provide important clues with respect to the physiology of those proteins in the oral cavity.

Role of hemocyte-derived granular components in invertebrate defense.

Figure 2 illustrates an outline of the cellular and humoral defense systems in limulus. On the basis of the knowledge described above, it is suggested that granular components present in L and S granules in the hemocytes play a decisive role in the biological defense for this animal. The isolated L granules contain at least three clotting factors plus coagulogen as the major component. The known anti-LPS factor and a number of additional unknown protein components are also present in the L granules. On the other hand, the isolated S granules contain antimicrobial tachyplesins as the major component, in addition to six unidentified proteins. We speculate that the L-granule-derived protein components, which probably contain all the factors essential for the Limulus clotting system participate, in immobilizing invading microbes, and that the S-granule-derived tachyplesins contribute to a self-defense system against invaders. Although we have not mentioned hemolymph plasma components, there are many humoral factors, such as proteinase inhibitors, alpha 2-macroglobulin, various lectins, C-reactive protein, and polyphemin, all of which are important for antimicrobial defense. Furthermore, Liu and colleagues have reported several endotoxin-binding proteins and a cell-adhesion protein found in the Limulus hemocytes. Although the exact functions of these substances are unknown, they may act in concert with other components to provide biological defense for the animal. Nevertheless, compared to our knowledge of mammalian blood cells, much less remains to be learned of biological/physiological events in horseshoe crab hemocytes.

Reducing citizen complaints

The multiple causes of complaints suggests that police administrators use a tailored approach to rehabilitating officers, rather than rely only on the traditional disciplinary and/or counselling strategies. Several principles seem appropriate in most cases: 1. Intervene as early as possible. 2. Use skill building strategies whenever possible (training is quicker and easier for officers to swallow than insight-based psychotherapy). 3. Be practical and job-related in designing programs (e.g.: Record and debrief specific calls rather than discuss patterns of communication) 4. Teach officers “situation processing’ skills so they can step outside their personal framework and become self-correcting. 5. Rely on supervisors and senior officers whenever possible so that new behaviors are reinforced immediately on the street. 6. Teach Verbal Judo as a basic self-defense system and also as a proven method of controlling citizens with negative attitudes. *** DIRECT SUPPORT *** A02CN017 00003

Cloning and characterization of a 23-kDa stress-induced mouse peritoneal macrophage protein.

Exposure of mouse peritoneal macrophages to oxidative and sulfhydryl-reactive agents in vitro enhances synthesis of a few cellular proteins that may be important in a self-defense system. A cDNA encoding a novel stress-inducible protein, designated MSP23 (macrophage 23-kDa stress protein), was cloned from a cDNA library of the macrophages by differential screening. A 1.0-kilobase mRNA transcript hybridized with the MSP23 cDNA gradually increased in macrophages upon culture in vitro. Treatment with diethylmaleate or glucose/glucose oxidase, which generates H2O2, markedly enhanced the induction of the transcript after several hours. Cadmium chloride and sodium arsenite also induced the transcript. An antiserum raised against recombinant MSP23 reacted with the 23-kDa stress-inducible protein of the macrophages. The amounts of 23-kDa protein in the cells rapidly increased during culture with diethylmaleate. The mRNA was detected in various tissues, and it was especially high in content in the liver. A search of databases revealed that six proteins of various species from bacteria to the mouse have a sequence homology to MSP23. One of the proteins is the C22 component of alkyl hydroperoxide reductase, which is induced by hydrogen peroxide in Salmonella typhimurium.

Separation of large and small granules from horseshoe crab (Tachypleus tridentatus) hemocytes and characterization of their components.

We designed a method for separating two types of granules, a smaller (S) but dense and a larger (L) but less dense granule from hemocytes of the horseshoe crab (Tachypleus tridentatus), using continuous sucrose density gradient centrifugation. The isolated L-granules contained at least three clotting factors plus a clottable protein, coagulogen, as the major component. The known anti-lipopolysaccharide factor and 7 additional unknown protein components were also present in the L-granules. Two known natural substrates, Pro-rich protein and 8.6 kDa protein, for limulus transglutaminase [Tokunaga, F., Yamada, M., Miyata, T., Ding, Y.-L., Hiranaga-Kawabata, M., Muta, T., Iwanaga, S., Ichinose, A., & Davie, E.W. (1993) J. Biol. Chem. 268, 252-261] were present in the L-granules. On the other hand, the isolated S-granules contained antimicrobial tachyplesins I and II (17 amino acids in length) as the major component, in addition to 6 unidentified proteins with molecular masses of less than 30 kDa. The structural analyses of tachyplesin analogs indicated that all these peptides of mature form are stored in the S-granules, together with a processing intermediate containing the COOH-terminal Gly-Lys sequence. We also found an Arg-rich protein of 22 kDa and a Leu-rich protein of 30 kDa in S-granules. Based on these observations, we speculate that protein components in L-granules, which probably contain all the factors essential for the limulus clotting system, participate in immobilization of invading microbes and that factors in the S-granules containing tachyplesins contribute to a self-defense system against invaders.

Formation and the inhibition of reaction plug in mated [formula omitted] — Study of a primitive defense reaction

Formation and inhibition of a reaction plug in the course of insemination reaction in Drosophila (Diptera) were investigated. When the reaction plug was formed in the vagina, fecundity was significantly reduced in interspecific crosses although the reaction plug did not affect the oviposition in intraspecific crosses. Some phenoloxidase blockers were found to be the effective inhibitors. The formation of the reaction plug was likely to be the consequence of polymerization and/or conformational change(s) of phenol-containing substance(s) involving nearly the same course of the melanization cascade reaction studied as a self-defense system in other insects.