Tissue and developmental expression of SRAM, an unconventional Rel-family protein.

Previously we have reported the purification and cDNA cloning of a novel Rel/Ankyrin-family protein named SRAM from the flesh fly, Sarcophaga peregrina. Rel proteins generally translocate into the nucleus upon immune stimuli by dissociating from an inhibitory ankyrin domain, while SRAM is unique in terms of its constitutive nuclear localization with its internal ankyrin domain accompanied, at least in a Sarcophaga cell line and fat body cells. Although SRAM had been originally identified as a sole factor that binds to the κB motif of the inducible Sarcophaga lectin gene promoter, its transcriptional activity remained controversial. Moreover, homologues of SRAM have not been found in any other established model organisms including Drosophila. Here we report that the developmental expression of SRAM was up-regulated at the early stages of embryogenesis and metamorphosis. Furthermore, SRAM expression was prominent in the digestive tracts of the third instar larvae. We argue the hypothesis that SRAM has evolved as a quite unconventional Rel-family protein in Sarcophaga.

Effect of 5-S-GAD on UV-B-induced cataracts in rats.

PURPOSE: 5-S-Glutathionyl-N-beta-alanyl-3,4-dihydroxyphenylalanine (5-S-GAD) is a novel antibacterial substance purified from Sarcophaga peregrina (flesh fly) that has both a radical scavenging activity and antioxidative activity. This is a report of an investigation of the effect of 5-S-GAD (eyedrops) on UVB-induced cataracts in rats. METHODS: Brown Norway male rats (n = 32; 7 weeks old) were treated with either 5-S-GAD 0.1%, 5-SGAD 1%, astaxanthin (AST) 0.1% suspension eyedrops or the vehicle alone (the solution without 5-S-GAD) three times a day (three doses at 5-min intervals each time). The treatment was scheduled 2 days before UV-B exposure and 2 days after UV-B exposure. Exposure to 100-200 mJ/cm(2) UV-B was performed once a week between drug treatments for 9 consecutive weeks, with a total dose of 1200 mJ/cm(2) UV-B. Ocular penetration of 5-S-GAD was analyzed using high-pressure liquid chromatography (HPLC). Cataract formation was documented by an anterior eye segment analysis system once a week under mydriasis. The light-scattering intensity (LSI) of the anterior superficial cortex region was measured. RESULTS: In the eighth to ninth week after the start of UV-B exposure, the LSI of anterior subcapsular lenses of 5-S-GAD-treated groups, as detected by HPLC, was significantly lower (P < 0.05) than that of the control, whereas no such difference was found in the AST-treated group. CONCLUSION: 5-S-GAD eyedrop application may delay the progression of UV-B-induced cataract in rats.

5-S-GAD, a novel radical scavenging compound, prevents lens opacity development.

The ability of N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD)-a novel catechol derivative isolated from an insect as an antibacterial substance-to scavenge free radicals and prevent cataract progression was examined. 5-S-GAD scavenged 1,1-diphenylpicrylhydrazyl (DPPH) and superoxide anions (O(2)(*)(-)), and inhibited lipid peroxidation. It also significantly inhibited the onset of glucocorticoid-induced lens opacification in chick embryos. These effects of 5-S-GAD were stronger than those of N-acetylcarnosine and TEMPOL, which are reported to be effective radical scavengers in the prevention of cataract progression. 5-S-GAD clearly delayed the maturation of cataracts induced by diamide in cultured lenses of rats. Daily instillation of 5-S-GAD retarded the development of lens opacity in galactose-fed rats. Biochemical analysis of the lenses revealed that 20-kDa proteins, presumably consisting of alpha-crystallin, were the most susceptible to oxidative stress, which leads to the carbonylation of the side chains of these proteins. alpha-Crystallin carbonylation induced by diamide or galactose was notably inhibited by 5-S-GAD in a dose-dependent manner. Our results show that 5-S-GAD prevents acute lens opacification in these short-term experimental models, possibly in part by virtue of its antioxidative property, and 5-S-GAD is expected to have long-term pharmaceutical effects.

The extracellular adenosine deaminase growth factor, ADGF/CECR1, plays a role in Xenopus embryogenesis via the adenosine/P1 receptor.

Adenosine deaminase-related growth factors (ADGF), also known as CECR1 in vertebrates, are a novel family of growth factors with sequence similarity to classical cellular adenosine deaminase. Although genes for ADGF/CECR1 have been identified in both invertebrates as well as vertebrates, their in vivo functions in vertebrates remain unknown. We isolated cDNA clones for two cerc 1s from Xenopus laevis. Both recombinant Xenopus CECR1s exhibited adenosine deaminase and growth factor activity, and the adenosine deaminase activity was found to be indispensable for growth factor activity. The Xenopus cerc 1s are expressed in the somites, pronephros, eyes, cement gland, neural tube, and neural floor plate of the embryos. Knock-down of these two genes using morpholino oligonucleotides caused a reduction in the body size and abnormalities of the body axis in the Xenopus embryos, accompanied by selective changes in the expression of developmental marker genes. Injection of adenosine, agonists for adenosine/P1 receptors, or adenosine deaminase inhibitor into late gastrula archenteron embryos resulted in developmental defects similar to those caused by morpholino oligonucleotide injection. These results show, for the first time, the involvement of CECR1s via the adenosine/P1 receptors in vertebrate embryogenesis via regulation of extracellular adenosine concentrations.

A long-lived o-semiquinone radical anion is formed from N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial substance.

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial peptide, generates hydrogen peroxide (H(2)O(2)) that exerts antitumour activity. We have investigated the precise mechanism of H(2)O(2) production from 5-S-GAD by autoxidation aiming to understand its action toward tumour cells. Using the electron spin resonance (ESR) technique, we detected a strong signal due to radical formation from 5-S-GAD. Surprisingly, the ESR signal of the radical derived from 5-S-GAD appeared after incubation for 30 min at 37 degrees C in the buffer at pH 7.4; the signal was persistently detected for 10 h in the absence of catalytic metal ions. The computer simulation of the observed ESR spectrum together with the theoretical calculation of the spin density of the radical species indicates that an o-semiquinone radical anion was formed from 5-S-GAD. We demonstrated that H(2)O(2) is produced via the formation of superoxide anion O2(.-) by the electron-transfer reduction of molecular oxygen by the 5-S-GAD anion, which is in equilibrium with 5-S-GAD in the aqueous solution. The radical formation and the subsequent H(2)O(2) production were inhibited by superoxide dismutase (SOD), when the antitumour activity of 5-S-GAD was inhibited by SOD. Thus, the formation of the o-semiquinone radical anion would be necessary for the antitumour activity of 5-S-GAD as an intermediate in the production of cytotoxic H(2)O(2).

Involvement of insect-derived growth factor (IDGF) in the cell growth of an embryonic cell line of flesh fly.

Insect-derived growth factor (IDGF) is the first adenosine deaminase-related growth factor (ADGF) purified from the conditioned medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Here we show the requirement of IDGF for the growth of NIH-Sape-4 cells. Growth factor activity was abolished by adsorption of IDGF from the conditioned medium of NIH-Sape-4 cells. In addition, knockdown of IDGF gene expression by RNA interference (RNAi) significantly reduced IDGF secretion from the cells following cell growth inhibition. The IDGF gene was strongly expressed in the hemocytes, and IDGF increased the viability of the larval hemocytes. These data provide evidence that IDGF is required for the growth of NIH-Sape-4 cells and possibly for hemocyte viability.

Inhibition of in vivo angiogenesis by N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine.

N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an antibacterial substance isolated from the flesh fly, inhibits human tumor growth in the nude mice model; however, the mechanism of its action is unclear. The in vivo antitumor effect includes the inhibition of tumor cell proliferation and suppression of angiogenesis. Angiogenesis is essential for tumor growth in vivo. In this study, we examined whether 5-S-GAD inhibits tumor cell-induced angiogenesis by performing the mouse dorsal air sac assay. We found that intraperitoneal administration of 5-S-GAD inhibited the angiogenesis induced by S180 mouse sarcoma cells. Furthermore, 5-S-GAD also inhibited vascular endothelial growth factor-induced angiogenesis in the Matrigel plug assay and embryonic angiogenesis in the chick embryo chorioallantoic membrane assay. However, 5-S-GAD did not show any effect on the proliferation, migration, and tube formation of vascular endothelial cells. These results provide the first evidence that a bioactive substance derived from the flesh fly has antiangiogenic activity in vivo, although the mechanisms involved could not be explained.

Transcription elongation factor S-II is required for definitive hematopoiesis.

Transcription elongation factor S-II/TFIIS promotes readthrough of transcriptional blocks by stimulating nascent RNA cleavage activity of RNA polymerase II in vitro. The biologic significance of S-II function in higher eukaryotes, however, remains unclear. To determine its role in mammalian development, we generated S-II-deficient mice through targeted gene disruption. Homozygous null mutants died at midgestation with marked pallor, suggesting severe anemia. S-II(-/-) embryos had a decreased number of definitive erythrocytes in the peripheral blood and disturbed erythroblast differentiation in fetal liver. There was a dramatic increase in apoptotic cells in S-II(-/-) fetal liver, which was consistent with a reduction in Bcl-x(L) gene expression. The presence of phenotypically defined hematopoietic stem cells and in vitro colony-forming hematopoietic progenitors in S-II(-/-) fetal liver indicates that S-II is dispensable for the generation and differentiation of hematopoietic stem cells. S-II-deficient fetal liver cells, however, exhibited a loss of long-term repopulating potential when transplanted into lethally irradiated adult mice, indicating that S-II deficiency causes an intrinsic defect in the self-renewal of hematopoietic stem cells. Thus, S-II has critical and nonredundant roles in definitive hematopoiesis.

Correlation between the catalase level in tumor cells and their sensitivity to N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD).

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD) exhibits selective cytotoxicity toward certain human tumor cell lines. 5-S-GAD has been shown to release hydrogen peroxide autonomously. Hydrogen peroxide is converted to water and oxygen by catalase. The purpose of this study is to determine whether or not 5-S-GAD exhibits selective cytotoxicity toward tumor cells with low catalase levels, but not toward ones with high catalase levels. We transfected MDA-MB-435S cells, which are sensitive to 5-S-GAD, with catalase cDNA to establish high catalase producer cells, and then examined their 5-S-GAD sensitivity. Similarly, we repressed catalase expression in T47D cells, which are insensitive to 5-S-GAD, by catalase RNA interference to create low catalase producer cells, and then examined their 5-S-GAD sensitivity. We show that the overexpression of catalase made MDA-MB-435S cells insensitive to 5-S-GAD, whereas the suppression of catalase made T47D cells sensitive to 5-S-GAD. The cellular catalase level was found to be crucial for cell sensitivity to 5-S-GAD.

Binding between azurocidin and calreticulin: its involvement in the activation of peripheral monocytes.

We found that azurocidin, a secretory protein in neutrophils, binds to calreticulin, a multifunctional chaperone of the endoplasmic reticulum. Azurocidin is known to induce cytokine production in monocytes, but the mechanism of monocyte activation by azurocidin remains unknown. On the other hand, an antibacterial peptide, KLKLLLLLKLK-NH(2) (L5), is known to bind to cell surface calreticulin of human neutrophils, resulting in their activation to produce O(2)(-). Therefore, we examined whether cell surface calreticulin is involved in the activation of human monocytes by azurocidin to produce IL-6. We found that carlreticulin is in fact located on the surface of monocytes and that the IL-6 production stimulated by an azurucidin is inhibited by anti-calreticulin antibody. Possibly, binding between cell surface calreticulin and azurocidin is prerequisite for the activation of monocytes by azurocidin to produce IL-6.

Participation of proteasomes in Xenopus embryogenesis.

We examined the effects of various protease substrates on Xenopus laevis embryogenesis. Thirty-three peptidyl-MCA substrates were added to the culture medium in which Xenopus embryos were developing. Five of the 33 substrates were found to inhibit embryogenesis at the early gastrula stage or much earlier ones. These results suggest that proteases that hydrolyze these substrates are involved in embryonic development. We found that the developmental stage of embryos is crucial for these substrates to inhibit their development. We purified a protease that hydrolyzes Pyr-Arg-Thr-Lys-Arg-MCA, a substrate that inhibits embryogenesis, from Xenopus embryos. This protease turned out to be a component of proteasomes. We found that 4 of the 5 substrates that inhibit embryogenesis are among the proteasome substrates. Thus, we concluded that proteasomes play a crucial role in the development of Xenopus embryos. Possibly, various catalytic subunits in proteasomes function independently, in stage-specific manners.

Involvement of H2O2 and O2- in the cytotoxicity of N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), a novel insect-derived anti-tumor compound.

We investigated the mechanism underlying the cytotoxicity of N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD) toward MDA-MB-435S, a human breast cancer cell line sensitive to 5-S-GAD. We found that the addition of either catalase or superoxide dismutase (SOD) to a culture medium of MDA-MB-435S cells almost completely abolished the cytotoxic effect of 5-S-GAD, indicating that both hydrogen peroxide (H(2)O(2)) and the superoxide anion (O(2)(-)) are involved in the cytotoxic action of 5-S-GAD. We compared the catalase and SOD levels in MDA-MB-435S and T47D, a cell line resistant to 5-S-GAD, and found that the levels in resistant cells are higher than those in sensitive cells. We concluded that the levels of these enzymes are crucial determinants of the sensitivity or insensitivity of cells to 5-S-GAD.

A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity.

Innate immunity is the first line of defence against infectious micro-organisms, and the basic mechanisms of pathogen recognition and response activation are evolutionarily conserved. In mammals, the innate immune response in combination with antigen-specific recognition is required for the activation of adaptive immunity. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Here, for the purpose of pharmaceutical screening, we established an in vitro culture based on the innate immune response of Drosophila. The in vitro system is capable of measuring lipopolysaccharide (LPS)-dependent activation of the immune deficiency (imd) pathway, which is similar to the tumour necrosis factor signalling pathway in mammals. Screening revealed that well-known inhibitors of phospholipase A(2) (PLA(2)), dexamethasone (Dex) and p-bromophenacyl bromide (BPB) inhibit LPS-dependent activation of the imd pathway. The inhibitory effects of Dex and BPB were suppressed by the addition of an excess of three (arachidonic acid, eicosapentaenoic acid and gamma-linolenic acid) of the fatty acids so far tested. Arachidonic acid, however, did not activate the imd pathway when used as the sole agonist. These findings indicate that PLA(2) participates in LPS-dependent activation of the imd pathway via the generation of arachidonic acid and other mediators, but requires additional signalling from LPS stimulation. Moreover, PLA(2) was activated in response to bacterial infection in Sarcophaga. These results suggest a functional link between the PLA(2)-generated fatty acid cascade and the LPS-stimulated imd pathway in insect immunity.

Activation of the Sarcophaga lectin gene promoter in transgenic Drosophila.

We established transgenic Drosophila strains in which the lacZ gene was expressed under the control of the 5'-upstream regulatory region of the Sarcophaga lectin gene promoter (3.1 kbp). The reporter gene was expressed in the fat bodies of the transgenic larvae when they were immunized by body pricking or treatment with Escherichia coli, which was the same as the Sarcophaga lectin gene expression in Sarcophaga larvae. However, the same reporter gene was found to be expressed constitutively in the digestive tracts of the transgenic larvae even without immunization.

Participation of two N-terminal residues in LPS-neutralizing activity of sarcotoxin IA.

Sarcotoxin IA is a cecropin-type antibacterial peptide of flesh fly. Using a mutant sarcotoxin IA lacking two N-terminal residues, we demonstrated that these residues are indispensable for its antibacterial activity against Escherichia coli and LPS-binding. Contrary to the native sarcotoxin IA, the mutant sarcotoxin IA could not neutralize various biological activities of LPS. It was suggested that sarcotoxin IA firmly binds to the lipid A core of LPS via these two N-terminal residues and forms a stable binding complex that exhibits no appreciable biological activity like native LPS.