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Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100-1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20-70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ogawa, Miura, Ikemoto, Ohnishi, Goto, Aoki, Motokurumada, Akimoto, Endo, Tsujimoto and Yanoshita.)

Background: Extracellular vesicles (EVs) have been isolated from various sources, including primary and cultured cell lines and body fluids. Previous studies, including those conducted in our laboratory, have reported the stability of EVs under various storage conditions., Methods: EVs from human whole saliva were separated via size-exclusion chromatography. To simulate the effects of gastric or intestinal fluids on the stability of EVs, pepsin or pancreatin was added to the samples. Additionally, to determine the effect of bile acids, sodium cholate was added. The samples were then subjected to western blotting, dynamic light scattering, and transmission electron microscopy analyses. In addition, the activity of dipeptidyl peptidase (DPP) IV retained in the samples was examined to monitor the stability of EVs., Results: Under acidic conditions, with pepsin mimicking the milieu of the stomach, the EVs remained stable. However, they partially lost their membrane integrity in the presence of pancreatin and sodium cholate, indicating that they may be destabilized after passing through the duodenum. Although several associated proteins, such as mucin 5B and CD9 were degraded, DPP IV was stable, and its activity was retained under the simulated gastrointestinal conditions., Conclusion: Our data indicate that although EVs can pass through the stomach without undergoing significant damage, they may be disrupted in the intestine to release their contents. The consistent delivery of active components such as DPP IV from EVs into the intestine might play a role in the efficient modulation of homeostasis of the signal transduction pathways occurring in the gastrointestinal tract., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (© 2021 The Author(s).)

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), is classified into two subgroups, Stx1 and Stx2. Clinical data clearly indicate that Stx2 is associated with more severe toxicity than Stx1, but the molecular mechanism underlying this difference is not fully understood. Here, we found that after being incorporated into target cells, Stx2, can be transported by recycling endosomes, as well as via the regular retrograde transport pathway. However, transport via recycling endosome did not occur with Stx1. We also found that Stx2 is actively released from cells in a receptor-recognizing B-subunit dependent manner. Part of the released Stx2 is associated with microvesicles, including exosome markers (referred to as exo-Stx2), whose origin is in the multivesicular bodies that formed from late/recycling endosomes. Finally, intravenous administration of exo-Stx2 to mice causes more lethality and tissue damage, especially severe renal dysfunction and tubular epithelial cell damage, compared to a free form of Stx2. Thus, the formation of exo-Stx2 might contribute to the severity of Stx2 in vivo, suggesting new therapeutic strategies against EHEC infections.

Macrophages secrete endoplasmic reticulum aminopeptidase 1 (ERAP1) in response to lipopolysaccharide (LPS) and interferon (IFN)-γ to enhance their phagocytic and nitric oxide (NO) synthetic activities. In this study, we found that a subset of secreted ERAP1 bound to exosomes released from LPS/IFN-γ-treated murine RAW264.7 macrophages compared to untreated cells. ERAP1-bound exosomes enhanced phagocytic and NO synthetic activities of macrophages more efficiently than free ERAP1 and exosomes derived from untreated cells. Deletion of the exon 10 coding sequence in ERAP1 gene resulted in loss of binding to exosomes. By comparing the activities of exosomes derived from wild-type and ERAP1 gene-deficient RAW264.7 cells, we observed that ERAP1 contributed to the exosome-dependent phagocytosis and NO synthesis of the cells. Upon stimulation of RAW264.7 cells with LPS/IFN-γ, TNF-α, IFN-γ, and CCL3 were also associated with the released exosomes. Analyses of cytokine function revealed that while CCL3 in the exosomes was crucial to the phagocytic activity of RAW264.7 cells, TNF-α and IFN-γ primarily contributed to the enhancement of NO synthesis. These results suggest that treatment with LPS/IFN-γ alters the physicochemical properties of exosomes released from macrophages in order to facilitate association with ERAP1 and several cytokines/chemokines. This leads to exosome-mediated enhancement of macrophage functions. It is possible that packaging effector molecules into exosomes upon inflammatory stimuli, facilitates the exertion of effective pathophysiological functions on macrophages. Our data provide the first evidence that ERAP1 associated with exosomes plays important roles in inflammatory processes via activation of macrophages., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nanomaterials are frequently used in microelectronics, cosmetics, and sunscreens. Platinum reagents are commonly used in disease diagnosis, cosmetics, and the food industry. Although research into the development of nanomaterialbased drug delivery systems has yielded promising results, the toxicity of these materials is not fully understood. We investigated the toxicity and drug interactions of 1- and 8-nm diameter platinum nanoparticles (nPt1 and nPt8, respectively) in mice. Acute hepato-renal toxicity of intravenously administered platinum nanoparticles was evaluated biochemically and histologically. Dose-dependent increases in serum markers of hepato-renal function (serum aminotransferases and blood urea nitrogen) were observed following administration of nPt1, whereas nPt8 had no effect, even at 20 mg/kg. Moreover, nPt1 induced interleukin (IL)-6 and IL-1β production 3 and 6 hours after administration. The effect of nPts on drug-induced toxicity was evaluated in mice injected intraperitoneally with carbon tetrachloride or cisplatin, with or without intravenous administration of platinum nanoparticles. All treatments in the absence of nanoparticles were non-lethal and resulted in moderate toxicity. However, exacerbated toxicity was observed in mice injected with carbon tetrachloride or cisplatin together with nPt1, but not in mice co-injected with nPt8. We found that nPt1 cause hepato-renal damage, and the effect is enhanced by chemical inducers of hepatotoxicity and nephrotoxicity. This is the first report demonstrating that nPt1 not only are hepatotoxic and nephrotoxic but also exacerbate drug toxicity. These findings will be useful for future nanotechnology and nanoscience research.

Exosomes are derived from various sources, including primary and cultured cell lines and body fluids. It is now evident that they are important for communication between cells. They have, therefore, been proposed as potential carriers to deliver drugs to specific sites. In this study, we examined stability of exosomes derived from human saliva. Exosomes were stored at 4°C for up to 20 months and their membrane integrity assessed. Several exosomal markers, such as dipeptidyl peptidase IV (DPP IV; membrane marker) and programmed cell death 6-interacting protein (Alix, lumen marker), were retained intact after 20 months storage at 4°C. Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release of Alix into the supernatant. In contrast, sodium dodecyl sulfate treatment caused a complete disruption of the membrane. In addition, membrane stability was maintained after freezing and thawing. These results indicated that human saliva-derived exosomes are stable, maintaining their membrane integrity over a long storage period.

Exosomes are small extracellular vesicles containing microRNAs and mRNAs that are produced by various types of cells. We previously used ultrafiltration and size-exclusion chromatography to isolate two types of human salivary exosomes (exosomes I, II) that are different in size and proteomes. We showed that salivary exosomes contain large repertoires of small RNAs. However, precise information regarding long RNAs in salivary exosomes has not been fully determined. In this study, we investigated the compositions of protein-coding RNAs (pcRNAs) and long non-protein-coding RNAs (lncRNAs) of exosome I, exosome II and whole saliva (WS) by next-generation sequencing technology. Although 11% of all RNAs were commonly detected among the three samples, the compositions of reads mapping to known RNAs were similar. The most abundant pcRNA is ribosomal RNA protein, and pcRNAs of some salivary proteins such as S100 calcium-binding protein A8 (protein S100-A8) were present in salivary exosomes. Interestingly, lncRNAs of pseudogenes (presumably, processed pseudogenes) were abundant in exosome I, exosome II and WS. Translationally controlled tumor protein gene, which plays an important role in cell proliferation, cell death and immune responses, was highly expressed as pcRNA and pseudogenes in salivary exosomes. Our results show that salivary exosomes contain various types of RNAs such as pseudogenes and small RNAs, and may mediate intercellular communication by transferring these RNAs to target cells as gene expression regulators.

Small non-coding RNAs, such as microRNAs (miRNAs), are involved in diverse processes, including organ development and tissue differentiation. Exosomes are small membrane vesicles (30-100 nm in diameter) produced by numerous cells. Recently, exosomes have been shown to contain miRNAs. However, the small RNAs contained in exosomes are not fully characterized. In a previous study, we found at least two types of salivary exosome that are different in size and have different proteomes. Studies of salivary exosomal small RNAs are limited to miRNAs. In this study, we examined small RNA transcriptomes using next generation sequencing technology to elucidate a full transcriptome set of small RNAs expressed in the two types of salivary exosomes and in whole saliva (WS). Many types of small RNA, such as miRNA, piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA) and other small RNAs are contained in salivary exosomes and WS. Among these small RNAs we identified novel miRNA candidates.

Saliva contains a large number of proteins that participate in the protection of oral tissue. Exosomes are small vesicles (30-100 nm in diameter) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We have recently demonstrated that exosomes are present in human whole saliva. In this study, we found that whole saliva contained at least two types of exosomes (exosome I and exosome II) that are different in size and protein composition. Proteomic analysis revealed that both types of exosomes contained Alix, Tsg101 and Hsp70, all exosomal markers, immunoglobulin A and polymeric immunoglobulin receptor, whereas they had different protein compositions. Most of dipeptidyl peptidase IV known as CD26 in whole saliva, was present on the exosome II and metabolically active in cleaving chemokines (CXCL11 and CXCL12). Human whole saliva exosomes might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

SSJ-127, a novel antimalarial rhodacyanine derivative, has shown potent antimalarial activity against chloroquine-resistant Plasmodium strains in vitro and subcutaneous administration of SSJ-127 results in a complete cure of a mouse malaria model. SSJ-127 was detected by fluorescence microscopy in the mouse malaria parasites Plasmodium berghei after exposure of infected red blood cells to the compound in vitro and in vivo. Selective accumulation of SSJ-127 in an organelle is observed in all blood stages of live malaria parasites. The organelle is clearly different from the mitochondrion and the nucleus in terms of morphology. The shape of the organelle changed during the asexual blood stages of the parasite. There was always a close association between the organelle and the mitochondrion. These results raised the possibility that SSJ-127 accumulates in an apicoplast of the malaria parasite and affects protozoan parasite-specific pathways., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Six allergic conjunctivitis patients (12 eyes) and 4 healthy volunteers (8 eyes) were investigated in terms of the effect of cooling sheets on eye itching and tear histamine concentration, before and 5 min after cooling the eyelids with cooling sheets. The severity of itching was evaluated with a five-level itching score. The combination treatment of levocabastine with cooling sheets significantly reduced eye itching, while no significant change in tear histamine concentration was observed before and after cooling sheet use. The cooling sheets are useful for reducing eye itching in the therapy of allergic conjunctivitis. The tear histamine concentration did not correlate with the antiitching effect of cooling sheets in this study.

A Gloydius blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences from known ecto-5'-nucleotidases (ecto-5'-NTs). Molecular cloning of ecto-5'-NT from G. blomhoffi brevicaudus venom predicted that it was a glycosyl phosphatidylinositol-anchored membrane protein containing 588 amino acid residues with 7 potential N-linked glycosylation sites. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian ecto-5'-NT sequences. This is the first report of the primary structure of ecto-5'-NT from a reptile. Gel-filtration chromatography of fresh venom from Gloydius blomhoffi blomhoffi, a subspecies of G. blomhoffi, revealed that at least a part of ecto-5'-NT is bound to exosome-like vesicles.

We have previously shown that maturation of mouse bone marrow-derived mast cells (BMMCs) into connective tissue mast cells (CTMCs) upon coculture with fibroblasts in the presence of stem cell factor (kit ligand) is accompanied by marked induction of a panel of genes, one of which was identified as NLRP3. Here we report that NLRP3 acts as a novel negative regulator of delayed prostaglandin (PG) D(2) production in BMMCs. We found that, apart from its cell maturation-associated induction, NLRP3 expression was markedly induced in BMMCs several hours after FcepsilonRI crosslinking or cytokine stimulation. Ectopic expression of NLRP3 in BMMCs resulted in marked attenuation of cyclooxygenase (COX)-2-dependent delayed PGD(2) generation, whereas it had no effects on other effector functions, including degranulation, COX-1-dependent immediate PGD(2) generation and cytokine/chemokine expression. The suppression of delayed PGD(2) generation by NLRP3 was preceded by a transient decrease of NF-kappaB activation and a marked reduction in the expression of COX-2, but not that of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), COX-1 and hematopoietic PGD(2) synthase. Moreover, in CTMC-like differentiated cells in which endogenous NLRP3 expression was induced, cytokine-stimulated induction of COX-2 and attendant delayed PGD(2) generation were markedly reduced. Our results suggest that, in mouse mast cells, NLRP3 counter-regulates COX-2-dependent sustained production of PGD(2), a prostanoid that exhibits both pro- and anti-allergic effects, thereby potentially influencing the duration of allergic and other mast cell-associated inflammatory diseases.

Saliva contains a large number of proteins that participate in the protection of oral tissue. We found, for the first time, small vesicles (30-130 nm in diameter) in human whole saliva. Vesicles from saliva were identified by electron microscopy after isolation by gel-filtration on Sepharose CL-4B. They resemble exosomes, which are vesicles with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We performed a biochemical characterization of these vesicles by amino acid sequence analysis and Western blot analysis. We found that they contain dipeptidyl peptidase IV (DPP IV), galectin-3 and immunoglobulin A, which have potential to influence immune response. The DPP IV in the vesicles was metabolically active in cleaving substance P and glucose-dependent insulinotropic polypeptide to release N-terminal dipeptides. Our results demonstrate that human whole saliva contains exosome-like vesicles; they might participate in the catabolism of bioactive peptides and play a regulatory role in local immune defense in the oral cavity.

Exosomes are small membrane vesicles (30-100 nm) with an endosome-derived limiting membrane that are secreted by a diverse range of cell types. We provide here the first evidence for the presence of exosome-like vesicles in snake venom. We isolated vesicles from fresh venom from Gloydius blomhoffii blomhoffii by gel-filtration. We found that the vesicles showed a typical exosome-like size and morphology as analyzed by electron microscopy. We observed that the vesicles contained dipeptidyl peptidase IV, aminopeptidase A, ecto-5'-nucleotidase and actin. Vesicle preparations truncated bioactive peptides such as angiotensin II, substance P, cholecystokinin-octapeptide, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. The role of these vesicles is still unknown, but they may affect blood pressure and glucose homeostasis following envenomation.

The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)(18)-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.

Dipeptidyl peptidase activity was investigated in snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis and Crotalus atrox. The strongest dipeptidyl peptidase IV (DPP IV) activity was found in venom from G. blomhoffi brevicaudus. The substrate specificity, susceptibility to inhibitors, and pH optimum of the partially purified enzyme were similar to those of known DPP IVs from bacteria and eukaryotes. The G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on amino acid sequences highly conserved in known DPP IVs. Two cDNA species encoding DPP IV were obtained, and designated as DPP IVa and DPP IVb. This is the first study to report the primary structure of DPP IV from a reptile. The deduced amino acid sequences for DPP IVa and DPP IVb both consist of 751amino acid residues and are highly homologous to each other. A putative catalytic triad for serine proteases, Ser-616, Asp-694, and His-726, is present. It is of particular interest that the deduced NH(2)-terminal sequence associated with the characteristic signal peptide is identical to that determined from the purified DPP IV. This indicates that the signal peptide of snake venom DPP IV is not cleaved off during biosynthesis, unlike those of other snake venom proteins.

The major lethal toxins present in the venoms of the red-headed krait, Bungarus flaviceps, and the Malayan krait, Bungarus candidus, have both been purified. Each consists of two polypeptide chains, A and B, joined by a disulfide bond. In the present study, primary structures of these toxins were determined by Edman degradation and by nucleotide sequencing of the cDNA clones. Amino acid sequencing of the N-terminus and enzymatically digested peptides revealed that the A and B chains were highly homologous to those of beta-bungarotoxins (beta-Bgts) from Bungarus multicinctus, respectively. We isolated cDNA clones encoding the A and B chains from both B. flaviceps and B. candidus venom gland cDNA libraries using probes designed based on the cDNA sequence of beta-Bgt from B. multicinctus. Two isoforms of the A chain and one isoform of the B chain were obtained from B. flaviceps, and one isoform of the A chain and two isoforms of the B chain were obtained from B. candidus. Both of the two A chains from B. flaviceps are made up of 119 amino acids and comprise 15 cysteine residues, while the A chains of beta-Bgt from other Bungarus species including B. candidus comprise 13 cysteine residues. The B chains from both species are composed of 59 amino acid residues and comprise seven cysteines. In conclusion, the lethal toxin from B. flaviceps is considered to be a novel isoform of beta-Bgt, which has a different pattern of cysteine residues from known beta-Bgts.

We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.

The sequence of a long-chain neurotoxin (71 amino acid residues, 10 half-cystines) from the venom of the African banded water cobra (Boulengerina annulata) was determined by Edman degradation. It exhibits high sequence similarity with long-chain neurotoxins from the venoms of four species of African cobras (genus Naja), which are collectively more similar to the Boulengerina toxin than to those of Asian Naja species. These results are discussed in the light of phylogenetic hypotheses on the relationships of African cobras.

N-Myc downregulated gene (NDRG) 1 is markedly induced during in vitro maturation of mouse immature bone marrow-derived mast cells (BMMCs) into a mature connective tissue mast cell (CTMC)-like phenotype. However, cellular function of this unique cytosolic protein is currently obscure. In this study, we sought potential NDRG1-binding proteins using yeast two-hybrid analysis and found that NDRG1 is capable of binding to heat-shock cognate protein 70 (Hsc70) both in vitro and in mast cells. The expression of Hsc70 was markedly elevated during the in vitro maturation of BMMCs into CTMC-like cells in accordance with the increased expression of NDRG1. Deletion of the C-terminal hydrophilic tandem repeats from NDRG1 facilitated the interaction with Hsc70 in vitro. Interaction between NDRG1 and Hsc70 was constitutive in mast cells and was not altered following cell activation. Although NDRG1 undergoes phosphorylation (accompanying paper), the binding of NDRG1 to Hsc70 was not affected by this event. Interestingly, the NDRG1-Hsc70 complex transiently appeared in the nuclear fraction of activated mast cells.

We previously demonstrated that the in vitro maturation of mouse immature bone marrow-derived mast cells into a mature connective tissue mast cell-like phenotype is accompanied by a marked induction of N-myc downregulated gene (NDRG) 1, a cytosolic protein with unknown function. Here we show that NDRG1 undergoes phosphorylation in mast cells. Recombinant NDRG1 was phosphorylated by calmodulin kinase-II, protein kinase (PK) A and PKC in vitro. Deletion of the C-terminal tandem repeats of NDRG1 resulted in increased phosphorylation by PKA and PKC, but not by calmodulin kinase-II. Furthermore, NDRG1 was phosphorylated on serine and threonine residues in mast cells, a process that was accelerated transiently following cell activation. Pharmacologic studies using kinase-specific inhibitors demonstrated that this NDRG1 phosphorylation in mast cells depended on calmodulin kinase-II and PKA, but not PKC. Collectively, our results indicate that NDRG1 is a multiphosphorylated protein in mast cells, and that the kinetics of increased NDRG1 phosphorylation parallels signaling events leading to exocytosis.

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.

We isolated the most lethal toxins in the venom of the Malayan krait (Bungarus candidus), one of the medically most important snake species in southeast Asia. Three beta-BTx like basic neurotoxins, T1-1, T1-2, and T2, with PLA2 activity were isolated from pooled venom of eight B. candidus from southern Thailand by cation-exchange chromatography, followed by adsorption chromatography on hydroxylapatite and RP-HPLC, with 14-, 16-, and 4-fold increases in toxicity compared to crude venom. The LDs50 determined in mice weighing 18-20 g were 0.26, 0.22, and 0.84 micro g per mouse with i.v. injection. T1-1 and T1-2 possessed comparable lethal toxicities to those of beta1-BTx, the most toxic neurotoxin in B. multicinctus venom, and the major neurotoxin in B. flaviceps venom. The apparent molecular weights of the native toxins were approximately 25-25.5 kDa. They consist of two polypeptide chains with apparent molecular weights of 15.5-16.5 and 8-8.5 kDa, respectively. The amino terminal sequences of the two chains of each of the toxins determined by Edman degradation exhibited considerable similarity with those of the A-chains and B-chains of beta-BTxs in the venom of Bungarus multicinctus.

Coculture of mouse bone marrow-derived mast cells (BMMC) with fibroblasts in the presence of stem cell factor (SCF) facilitates morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype. By means of cDNA subtraction, we identified several inducible genes during this mast cell maturation process. Of approximately 100 sequenced clones induced, nearly 50% were chromosome 14-associated serine proteases. Approximately 14% encoded NDRG1, a 43-kDa cytosolic protein that has been implicated in cell differentiation. NDRG1 was distributed in the cytosol of cultured mast cells and CTMC in rat skin. Overexpression of NDRG1 in RBL-2H3 cells resulted in enhanced degranulation in response to various stimuli. Thus, NDRG1 may be a mast cell maturation-associated inducible protein that allows the cells to be susceptible to extracellular stimuli leading to degranulation. Additionally, several unique maturation-associated inducible genes were identified, molecular and functional characterization of which will provide new insights into mast cell biology.

Blomhotin is a novel peptide (pGlu1-Gly2-Arg3-Pro4-Pro5-Gly6-Pro7-Pro8-Ile9-Pro10-Arg11) which has been isolated from the venom of Agkistrodon halys blomhoffii and exhibits contractile activity on rat stomach fundus. We carried out a structure-activity study of blomhotin and its related peptides, and the findings suggested that the N-terminal portion of blomhotin is mainly responsible for affinity for the blomhotin receptor, whereas the C-terminal portion of blomhotin, Pro-Ile-Pro-Arg, is responsible for complete activation of the blomhotin receptor in the rat stomach fundus. In particular, the amino acids at positions 9 and 11 of blomhotin appear to be essential for binding and intrinsic activity. Using knowledge gained from this structure-activity analysis, we have identified photoactive blomhotin analogues that have sufficient biological activity to probe the target molecule of blomhotin.

A cDNA clone, 1.8 kb long, was isolated from a venom gland cDNA library of Agkistrodon blomhoffi that encodes a large plurifunctional precursor composed of 263 amino-acid residues. Nucleotide sequence analysis of this clone revealed that sequences which code for blomhotin and a novel peptide Leu3-blomhotin are located in the N-terminal region of the precursor polypeptide, followed by four tandemly aligned sequences which code for three types of bradykinin-potentiating peptide. In the C-terminal region, the sequence for the C-type natriuretic peptide was located along with a preceding processing signal. The deduced amino-acid sequences for the four bradykinin-potentiating peptides coincided exactly with previously known sequences for potentiator B, potentiator C and potentiator E. The actual Leu3-blomhotin peptide was subsequently isolated from the venom of A. blomhoffi and characterized. Leu3-blomhotin possesses contractile activity in isolated rat stomach fundus smooth muscle in the same manner as blomhotin. Furthermore, it was shown that blomhotin and Leu3-blomhotin retained activity to inhibit the angiotensin-converting enzyme.

We have previously observed that all human hepatocellular carcinomas (HCCs) from HBV carriers examined had the integrated X region. In this study, HBV DNA was isolated from an integration site in one HCC that had a single, very small integrated viral DNA including the X region, but it had no expression of X gene as poly(A)RNA. It was found that HBV DNA was present between alphoid repetitive sequences, and it included Enhancer and X regions, encompassing the adr sequence from 910 to 1811. Nucleotides for 8 amino acids at the 3' end, a stop codon of X gene and a poly(A) signal downstream of X gene were lost by integration, and nucleotides for 7 amino acids and a stop codon were substituted by a connected alphoid sequence. When this cloned HBV DNA was transfected with an expression vector to an immortalized mouse liver epithelial cell line, MLE-10, malignant transformation occurred. Transformants having expressed poly(A)RNA of the X gene showed anchorage-independent growth in soft agar and tumor formation in the subcutis of nude mice. The mRNA level of EGFR was found to be remarkably enhanced in X-transformed cells, in contrast with the absence of this mRNA in parental and ras-transformed MLE-10. Our data provide evidence that the Enhancer-X region alone is the key contributor to the malignant change of pre-malignant liver cells in HBV carriers through activation of some specific genes, such as EGFR., (Copyright 2000 Wiley-Liss, Inc.)

Licorice root traditionally used as an anti-inflammatory drug exhibited an inhibitory effect on lysoPAF (platelet-activating factor) acetyltransferase in vitro: the ether soluble fraction of the crude drug produced a 27.3% inhibition at a concentration 10 microg/ml. From this fraction, licoricidin (1), 1-methoxyphaseollin (2), 6,8-diprenylgenistein (3) and 1-methoxyphaseollidin (4) were isolated as active components, whose IC50 values were 7.7, 57, 19 and 48 microM, respectively. Licoricidin (1) seems to be one of the most potent compounds of plant origin isolated so far.

The mechanisms of non-drowsiness after oral administration of TMK688 (1- [[5'-(3"-methoxy-4"-ethoxycarbonyloxyphenyl)-2',4'-pentadienoyl ] aminoethyl]-4-diphenylmethoxypiperidine, CAS 110501-66-1) were investigated using mice. TMK688 inhibited the histamine-induced vascular permeability at oral doses of 3.2-10 mg/kg with an ID50 value of 5.4 mg/kg. More than 100 times higher doses were needed to prolong the hexobarbital-induced sleeping. Pyrilamine, a typical antihistamine agent, showed little difference among these doses and antiallergic drugs having antihistamine activity, i.e., terfenadine, azelastine and ketotifen, had effects between TMK688 and pyrilamine. The inhibitory activity of orally administered TMK688 against ex vivo [3H]-pyrilamine binding to mouse cerebral histamine receptors appeared at the same doses as its potentiating activity against hexobarbital-induced sleeping. When given orally, TMK688 was hydrolyzed to TMK777 (CAS 101619-11-8), then conjugated with glucuronic acid to TMK777-glucuronide. No TMK688 was detected in the blood. The main metabolite TMK777-glucuronide could hardly penetrate the blood-brain barrier because of its polarity. Although the plasma concentrations of TMK777 were far lower than those of TMK777-glucuronide, TMK777 was penetrable into the brain and the cerebral concentrations of TMK777 increased in parallel with the plasma concentrations of the drug. Since intracerebroventricularly-injected TMK777 prolonged the sleeping time, and since the threshold concentration of TMK777 in the cerebral cortex to potentiate the hexobarbital-induced sleeping was consistent despite different administration routes, the drowsiness elicited by markedly high doses of TMK688 is though to be caused by intracerebral TMK777. In other words, TMK688 does not seem to cause drowsiness at effective doses because of the poor prenetrability of its main metabolites into the brain.

Turcot syndrome is characterized by an association of malignant brain tumors and colon cancer developing in the patient's teens. Since the mechanism of carcinogenesis in Turcot syndrome is still unclear, we analysed genetic changes in tumors from a Turcot patient with no family history of the condition. All tumors, including one astrocytoma, three colon carcinomas, and two colon adenomas, exhibited severe replication error (RER), and all colon tumors showed somatic mutations at repeated regions of TGFbetaRII, E2F-4, hMSH3, and/or hMSH6 genes. Somatic APC mutations were detected in three of three colon carcinomas, and somatic p53 mutations were detected in the astrocytoma and two of three colon carcinomas, both of which showed two mutations without allele loss. We also found that normal colon mucosa, normal skin fibroblasts and normal brain tissue from this patient showed respective high frequencies of RER, in contrast to usual HNPCC patients in which RER was very rare in normal tissues. These results suggest that extreme DNA instability in normal tissues causes the early development of multiple cancer in Turcot syndrome. A missense mutation (GAG to AAG) at codon 705 of hPMS2 gene was detected in one allele of this patient, which was inherited from his mother without tumors. Additional unknown germline mutation may contribute to the genetic instability in normal tissues.

The significance of CD44 variants in gastric carcinoma has not been fully investigated in terms of the pathological features of the carcinoma, including its association with Epstein-Barr virus (EBV). In this study, a total of 104 primary gastric carcinoma tissues (EBV-associated gastric carcinomas, EBVaGC, and EBV-negative carcinomas) were evaluated by immunohistochemistry. When the immunoreactivity of formalin-fixed, paraffin-embedded sections was graded on a scale of 0-3, the frequencies of grades 0-1, 2 and 3 were, respectively, 77%, 16% and 7% using monoclonal antibody (MAb) 3G5, which recognizes V3-5, and 70%, 14% and 15% with MAb 2F10, which recognizes V6. The expression of CD44 variants is independently correlated with lymph node metastasis and EBV-association in gastric carcinoma. Significant correlations were observed between V3-5 expression and lymph vessel invasion or lymph node metastasis, and between V6 expression and lymph node metastasis. The expression of both variants was significantly correlated with EBV-association. EBV-association and lymph node metastasis contributed independently to CD44 variant expression by multivariate analysis. Thus, the mechanism and significance of CD44 variant-expression are different in gastric carcinomas with or without EBV. EBVaGC is a distinct type of gastric carcinoma which should be considered separately from EBV-negative carcinoma.

Colorectal tumors frequently have loss of heterozygosity on chromosome 22q, suggesting that inactivation of tumor suppressor gene(s) on 22q participates in the tumor development. Neurofibromatosis 2 (NF2) gene and E1A binding protein p300 gene, recently identified on 22q, are thought to be candidates for tumor suppressor genes. In this study, mutation of the NF2 gene in 59 colorectal carcinomas, and mutation of the p300 gene in 27 colorectal and two gastric carcinomas, were analysed using PCR-SSCP, RT-PCR-SSCP and direct sequencing methods. Missense mutations of p300 gene were detected in a colorectal carcinoma, and in a gastric carcinoma, though no mutation of NF2 gene was detected. Both p300 mutations were somatic and coupled to deletion of the second allele of the gene, which suggests inactivation of the p300 gene, in these carcinomas. The mutations are located within the Cys/His-rich regions, which are assumed to play important roles in the function of p300. These are the first cases in which p300 gene has been found to be altered in both alleles, suggesting that inactivation of the p300 gene may be involved in the development of carcinomas, and that this gene may be the target of loss of 22q in carcinomas of the digestive tract.

The development of human colon carcinomas is associated with a number of genetic alterations. A high frequency of deletion of the short arm of chromosome 8 at a late stage of colon carcinogenesis was detected by DNA analysis of colon carcinomas, which suggests the presence of a tumor suppressor gene. We therefore, introduced normal human chromosome 8 into colon carcinoma cells that showed allele loss on 8p21, through microcell hybridization. Five clones of hybrid cells were obtained from independent experiments. Three hybrids exhibited morphological alteration and suppressed tumorigenicity in the subcutis of nude mice, but the other two did not. The difference between the two types of hybrids was the region of the introduced normal chromosome 8: Three hybrids exhibiting morphological alteration and suppressed tumorigenicity had the entire region of the introduced chromosome 8, whereas the other two, exhibiting no change, lacked 8p12-pter from the introduced chromosome. Furthermore, the invasiveness of the hybrids with suppressed tumorigenicity was reduced to one-fifth of that of the parental cells. These results indicate that 8p12-pter carries a gene that contributes to suppression of both tumorigenicity and invasiveness of colon carcinomas.

Metastasis of colon carcinomas is assumed to be caused by multiple steps, which include a loss of cell adhesion that results in the release of carcinoma cells from the original tumor tissue. A human colon carcinoma cell line COKFu was established from a poorly differentiated metastatic adenocarcinoma without cell-cell adhesion and without expression of E-cadherin mRNA and protein. This cell line was co-transfected with mouse E-cadherin cDNA in an expression vector and a neomycin-resistant gene. The parental carcinoma cells had a spindle shape and were scattered, whereas the transfected cells, which expressed exogenous E-cadherin gene, showed a more compact shape with strong cell-cell adhesion and with increased adhesiveness to collagen gel. These cells showed a significantly low anchorage independency (2-7%) and decreased invasiveness (30%) compared to the parental cells. Growth rate of transfectants was decreased both in vitro and in the subcutis of nude mice, with decreased lymphnode metastasis in the case of intravenous injection. It was additionally found that activity of 62 kd gelatinase, secreted from parental cells, was lost or decreased in E-cadherin-transfected cells. These results suggest that E-cadherin is not only involved in the cell-cell adhesion of colon carcinomas, it also has a wider effect, including cell-substratum adhesion and the regulation of proteinase secretion from the cells, resulting in partial suppression of invasiveness and tumorigenic growth.

Eleven different missense and one nonsense mutant-type p53 cDNAs, which have been frequently detected in human colorectal carcinomas, were constructed and examined for their ability to cooperate with activated human H-ras genes, pSK2 and pHs49, in transfection of rat embryo fibroblasts (REF). Each missense mutant-type p53 cDNA with either of the two activated H-ras genes transformed REF with a different frequency of transformation depending on the different kind of mutation, whereas wild-type p53 (with ras), nonsense mutant-type p53 (with ras), as well as mutant-type p53 (alone) and ras (alone), did not transform REF. Six transformed REF cell lines were established from cotransfection with missense mutant-type p53 cDNA and ras gene; all of them exhibiting exogenous human p53 DNA, RNA, protein, and H-ras DNA and RNA. All six transformed cell lines showed both tumorigenicity and lung metastatic potential in nude mice. They also exhibited 92 kilodalton gelatinase activity, which was not detected in parental REF. These results suggest that missense mutations in p53 gene have a role in malignant transformation as well as metastatic potential.

We analyzed somatic mutations of the adenomatous polyposis coli (APC), p53, and K-ras genes in gastroduodenal polyps and normal gastroduodenal mucosa from 21 familial adenomatous polyposis patients, using PCR-single-strand conformation polymorphism and direct sequencing methods. Seventy-five polyps were obtained from these patients endoscopically or surgically, and they were histopathologically diagnosed as mild adenoma, moderate adenoma, severe adenoma, adenocarcinoma, and fundic gland polyp. Examining the APC-coding region where somatic mutations in colorectal tumors are known to be clustered, we detected 47 somatic mutations. The frequency of mutation detected was 6 of 9 (67%) in ampullary adenomas, 1 of 2 (50%) in ampullary adenocarcinoma, 11 of 24 (46%) in non-ampullary adenomas, 26 of 29 (90%) in gastric adenomas, and 3 of 11 (27%) in gastric fundic gland polyps. These mutations frequently occurred at codons 1450, 1462-1465, and 1554-1556, the third being a newly found hot spot. All mutations formed stop codons that resulted in truncated APC proteins. K-ras mutation was detected only in an ampullary adenocarcinoma, and p53 mutation was not detected in any of the tumors analyzed. There was no somatic mutation detected in samples of flat mucosa that were diagnosed as normal mucosa both endoscopically and histopathologically. Frequent APC mutations in mild and small adenomas, similar to the findings in severe and large adenomas, suggested that the genetic change in the APC gene occurs in an early stage of forming gastroduodenal adenomas. Moreover, the presence of somatic APC mutations in fundic gland polyps suggests that inactivation of the APC gene plays a role not only in forming adenomas but also in forming hyperplastic polyps in fundic gland mucosa, and there may be some additional steps to the adenoma-carcinoma sequence.