Distribution of tetrodotoxin in the xanthid crab (Atergatis floridus) collected in the coastal waters of Kanagawa and Wakayama Prefectures

The objective of this investigation was to study the distribution of tetrodotoxin (TTX) in the xanthid crab (Atergatis floridus) found in the coastal waters of Kanagawa and Wakayama Prefectures of Japan using mouse assay methods. We used 32 crab samples (18 males and 14 females) and toxicity was analyzed on 13 parts of the body of each sample. The muscle of chelipeds was found to be toxic in all the samples with a wide range of toxicity (5–237 MU/g), whereas the toxicity in the muscle of the cephalothorax was found to be non-toxic (below detectable limit) in all the samples [Narita, H., Watanabe, K., Baba, K., Ohgami, H., Ai, T.K., Igarashi, Y., Nara, M., Noguchi, T., Hashimoto, K., 1987. The toxicity of digestive gland of trampet shells inhabiting the coast of Shizuoka Prefecture. J. Food Hyg. Soc. Jpn. 28, 115–118.]. Further investigation of different parts of the chelipeds indicated that the muscle of the palm and carpus are usually toxic and that of merus and ischium are almost non-toxic. Toxicity of the muscles of palm ranged between 7 and 52 MU/g, whereas toxicity of the muscle of ischium was below detectable limit. Results from our study indicate clear contrast in the distribution of tetrodotoxin in muscles of different parts of the xanthid crabs, plausibly due to some inherent physiological mechanism. Further investigation is necessary to understand the mechanism responsible for such contrast.     

β-Naphthoflavone analogs as potent and soluble aryl hydrocarbon receptor agonists: Improvement of solubility by disruption of molecular planarity

The physiological role of aryl hydrocarbon receptor (AhR) is not yet fully understood, and investigation is hampered by the limited solubility of reported AhR ligands in aqueous media. To achieve improved solubility, we focused on our previous finding that planarity-disruption of molecules leads to less efficient crystal packing and greater aqueous solubility. Here, we describe chemical modification of an AhR agonist, β-naphthoflavone, focusing on planarity-disruption. As expected, introduction of substituents at the ortho-positions of the phenyl group resulted in greater solubility. Among the compounds prepared, the fluoro analog showed more potent AhR agonistic activity and greater solubility than did β-naphthoflavone. Our results indicate that this strategy to improve aqueous solubility, that is, introduction of substituent(s) that disrupt planarity, may be generally applicable to bicyclic molecules.     

The trehalose transporter 1 gene sequence is conserved in insects and encodes proteins with different kinetic properties involved in trehalose import into peripheral tissues

We recently cloned a trehalose transporter gene (Tret1) that contributes to anhydrobiosis induction in the sleeping chironomid Polypedilum vanderplanki Hinton. Because trehalose is the main haemolymph sugar in most insects, they might possess Tret1 orthologs involved in maintaining haemolymph trehalose levels. We cloned Tret1 orthologs from four species in three insect orders. The similarities of the amino acid sequence to TRET1 in P. vanderplanki were 58.5–80.4%. Phylogenetic analysis suggested the Tret1 sequences were conserved in insects. The Xenopus oocyte expression system showed apparent differences in the K_m and V_max values for trehalose transport activity among the six proteins encoded by the corresponding orthologs. The TRET1 orthologs of Anopheles gambiae (K_m: 45.74 ± 3.58 mM) and Bombyx mori (71.58 ± 6.45 mM) showed low trehalose affinity, whereas those of Apis mellifera (9.42 ± 2.37 mM) and Drosophila melanogaster (10.94 ± 7.70 mM) showed high affinity. This difference in kinetics might be reflected in the haemolymph trehalose:glucose ratio of each species. Tret1 was expressed not only in the fat body but also in muscle and testis. These findings suggest that insect TRET1 is responsible for the release of trehalose from the fat body and the incorporation of trehalose into other tissues that require a carbon source, thereby regulating trehalose levels in the haemolymph.     

Analyzing steady states of dynamics of bio-molecules from the structure of regulatory networks

Regulatory relations between biological molecules constitute complex network systems and realize diverse biological functions through the dynamics of molecular activities. However, we currently have very little understanding of the relationship between the structure of a regulatory network and its dynamical properties. In this paper we introduce a new method, named “linkage logic” to analyze the dynamics of network systems. By this method, we can restrict possible steady states of a given complex network system from the knowledge of regulatory linkages alone. The regulatory linkage simply specifies the list of variables that affect the dynamics of each variable. We formalize two aspects of the linkage logic: the “Principle of Compatibility” determines the upper limit of the diversity of possible steady states of the dynamics realized by a given network; the “Principle of Dependency” determines the possible combinations of states of the system. By combining these two aspects, (i) for a given network, we can identify a cluster of nodes that gives an alternative representation of the steady states of the whole system, (ii) we can reduce a given complex network into a simpler one without loss of the ability to generate the diversity of steady states, (iii) we can examine the consistency between the structure of network and observed set of steady states, and (iv) sometimes we can predict unknown states or unknown regulations from an observed set of steady states alone. We illustrate the method by several applications to an experimentally determined regulatory network for biological functions.     

Pathogenesis of Hepatitis C Virus Infection in Tupaia belangeri

The lack of a small-animal model has hampered the analysis of hepatitis C virus (HCV) pathogenesis. The tupaia (Tupaia belangeri), a tree shrew, has shown susceptibility to HCV infection and has been considered a possible candidate for a small experimental model of HCV infection. However, a longitudinal analysis of HCV-infected tupaias has yet to be described. Here, we provide an analysis of HCV pathogenesis during the course of infection in tupaias over a 3-year period. The animals were inoculated with hepatitis C patient serum HCR6 or viral particles reconstituted from full-length cDNA. In either case, inoculation caused mild hepatitis and intermittent viremia during the acute phase of infection. Histological analysis of infected livers revealed that HCV caused chronic hepatitis that worsened in a time-dependent manner. Liver steatosis, cirrhotic nodules, and accompanying tumorigenesis were also detected. To examine whether infectious virus particles were produced in tupaia livers, naive animals were inoculated with sera from HCV-infected tupaias, which had been confirmed positive for HCV RNA. As a result, the recipient animals also displayed mild hepatitis and intermittent viremia. Quasispecies were also observed in the NS5A region, signaling phylogenic lineage from the original inoculating sequence. Taken together, these data suggest that the tupaia is a practical animal model for experimental studies of HCV infection.Hepatitis C virus (HCV) is a small enveloped virus that causes chronic hepatitis worldwide (32). HCV belongs to the genus Hepacivirus of the family Flaviviridae. Its genome comprises 9.6 kb of single-stranded RNA of positive polarity flanked by highly conserved untranslated regions at both the 5′ and 3′ ends (4, 27, 29). The 5′ untranslated region harbors an internal ribosomal entry site (29) that initiates translation of a single open reading frame encoding a large polyprotein comprising about 3,010 amino acids (35). The encoded polyprotein is co- and posttranslationally processed into 10 individual viral proteins (15).In most cases of human infection, HCV is highly potent and establishes lifelong persistent infection, which progressively leads to chronic hepatitis, liver steatosis, cirrhosis, and hepatocellular carcinoma (9, 16, 21). The most effective therapy for treatment of HCV infection is administration of pegylated interferon combined with ribavirin. However, the combination therapy is an arduous regimen for patients; furthermore, HCV genotype 1b does not respond efficiently (19). The prevailing scientific opinion is that a more viable option than interferon treatment is needed.The chimpanzee is the only validated animal model for in vivo studies of HCV infection, and it is capable of reproducing most aspects of human infection (5, 18, 23, 28, 35, 36). The chimpanzee is also the only validated animal for testing the authenticity and infectivity of cloned viral sequences (8, 14, 35, 36). However, chimpanzees are relatively rare and expensive experimental subjects. Cross-species transmission from infected chimpanzees to other nonhuman primates has been tested but has proven unsuccessful for all species evaluated (1).The tupaia (Tupaia belangeri), a tree shrew, is a small nonprimate mammal indigenous to certain areas of Southeast Asia (6). It is susceptible to infection with a wide range of human-pathogenic viruses, including hepatitis B viruses (13, 20, 31), and appears to be permissive for HCV infection (33, 34). In an initial report, approximately one-third of inoculated animals exhibited acute, transient infection, although none developed the high-titer sustained viremia characteristic of infection in humans and chimpanzees (33). The short duration of follow-up precluded any observation of liver pathology. In addition to the putative in vivo model, cultured primary hepatocytes from tupaias can be infected with HCV, leading to de novo synthesis of HCV RNA (37). These reports strongly support tupaias as a valid model for experimental studies of HCV infection. However, longitudinal analyses evaluating the clinical development and pathology of HCV-infected tupaias have yet to be examined. In the present study, we describe the clinical development and pathology of HCV-infected tupaias over an approximately 3-year time course.     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.     

Structural Basis for Conformational Dynamics of GTP-bound Ras Protein

Ras family small GTPases assume two interconverting conformations, “inactive” state 1 and “active” state 2, in their GTP-bound forms. Here, to clarify the mechanism of state transition, we have carried out x-ray crystal structure analyses of a series of mutant H-Ras and M-Ras in complex with guanosine 5′-(β,γ-imido)triphosphate (GppNHp), representing various intermediate states of the transition. Crystallization of H-RasT35S-GppNHp enables us to solve the first complete tertiary structure of H-Ras state 1 possessing two surface pockets unseen in the state 2 or H-Ras-GDP structure. Moreover, determination of the two distinct crystal structures of H-RasT35S-GppNHp, showing prominent polysterism in the switch I and switch II regions, reveals a pivotal role of the guanine nucleotide-mediated interaction between the two switch regions and its rearrangement by a nucleotide positional change in the state 2 to state 1 transition. Furthermore, the ³¹P NMR spectra and crystal structures of the GppNHp-bound forms of M-Ras mutants, carrying various H-Ras-type amino acid substitutions, also reveal the existence of a surface pocket in state 1 and support a similar mechanism based on the nucleotide-mediated interaction and its rearrangement in the state 1 to state 2 transition. Intriguingly, the conformational changes accompanying the state transition mimic those that occurred upon GDP/GTP exchange, indicating a common mechanistic basis inherent in the high flexibility of the switch regions. Collectively, these results clarify the structural features distinguishing the two states and provide new insights into the molecular basis for the state transition of Ras protein.