In this study, we investigated the carcinogenic response of transgenic mice carrying the human prototype c-Ha-ras gene, namely Tg rasH2/CB6F1 mice, to various genotoxic carcinogens and compared it with that of control non-transgenic CB6F1 mice (non-Tg mice). The present studies were conducted as the first step in the evaluation of the Tg rasH2/CB6F1 mouse as a model for the rapid carcinogenicity testing system. Short-term (< or = 6 months) rapid carcinogenicity tests of various genotoxic carcinogens, 4-nitroquinoline-1-oxide, cyclophosphamide, N,N-diethylnitrosamine, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol, revealed that Tg rasH2/CB6F1 mice are more susceptible to these genotoxic carcinogens than control non-Tg mice. Tg rasH2/CB6F1 mice developed tumors more rapidly compared with non-Tg mice. Malignant tumors were observed only in the carcinogen-treated Tg rasH2/CB6F1 mice, but not in non-Tg mice treated with the same carcinogens. Each carcinogen induced tumors in corresponding target tissues of the Tg rasH2/CB6F1 mice. Only a very few lung adenomas but no other tumors were seen as spontaneous tumors during the 6 months of carcinogenicity tests. These results demonstrate that more rapid onset and higher incidence of more malignant tumors can be expected with high probability after treatment with various genotoxic carcinogens in the Tg rasH2/CB6F1 mice than in control non-Tg mice. The Tg rasH2/CB6F1 mouse seems to be a promising candidate as an animal model for the development of a rapid carcinogenicity testing system.

It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WT-FUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions.

Peutz-Jeghers syndrome (PJS) is a dominantly inherited human disorder characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. LKB1 (STK11) serine͞threonine kinase is the product of the causative gene of PJS, which has been mapped to chromosome 19p13.3. However, several studies have produced results that are not consistent with a link between LKB1 gene mutation and PJS. We constructed a knockout gene mutation of Lkb1 to determine whether it is the causative gene of PJS and to examine the biological role of the Lkb1 gene. Lkb1 ؊/؊ mice died in utero between 8.5 and 9.5 days postcoitum. At 9.0 days postcoitum, Lkb1 ؊/؊ embryos were generally smaller than their age-matched littermates, showed developmental retardation, and did not undergo embryonic turning. Multiple gastric adenomatous polyps were observed in 10-to 14-month-old Lkb1 ؉/؊ mice. Our results indicate that functional Lkb1 is required for normal embryogenesis and that it is related to tumor development. The Lkb1 ؉/؊ mouse is suitable for studying molecular mechanism underlying the development of inherited gastric tumors in PJS.tumor ͉ embryo development

N/A. Laryngoscope, 126:E233-E239, 2016.

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IntroductionFactor VIII (FVIII) is a glycoprotein cofactor that serves as a critical component in the intrinsic coagulation pathway. 1 Insufficient expression of FVIII or expression of nonfunctional FVIII results in hemophilia A, one of the most common severe hereditary bleeding disorders. Mature FVIII is synthesized as a single-chain polypeptide consisting of 2332 amino acid residues. 2,3 Based on internal sequence homologies, 3 types of structural domains have been identified, arranged in the order of A1-A2-B-A3-C1-C2. 4 FVIII circulates in plasma as a heterodimer of a heavy chain, consisting of the A1 (1-372) and A2 (373-740) domains, together with heterogeneous fragments of partially proteolyzed B domains , and a light chain (LCh) consisting of the A3 (1649-2019), C1 (2020-2172), and C2 (2173-2332) domains. 3,4 Thrombin and factor Xa (FXa) are the major physiologic enzymes that activate FVIII. Both enzymes cleave FVIII at Arg372, Arg740, and Arg1689 and transform it to its active form, an A1/A2/A3-C1-C2 heterotrimer. 5 Activated FVIII is incorporated into the FXase complex assembled on a phospholipid (PL) membrane surface and thus acts as an accelerator of FXa generation. Activated protein C (APC) inactivates activated FVIII by proteolytic cleavage at Arg336 in the presence of negatively charged PL. 5 In this reaction, the 54-kd A1 fragment is cleaved into a 40-kd fragment with a loss of the carboxy-terminal acidic region. The APC binding site in the FVIII molecule has been located within the residues 2009 to 2018 in the A3 domain of the LCh. 6 The site has not been confirmed in direct binding experiments, however.FVIII circulates in plasma as a noncovalent complex with von Willebrand factor (VWF). 7 VWF regulates the synthesis and cofactor activity of FVIII. 8 Furthermore, VWF concentrates FVIII at the site of vascular injury. 9 The absence of VWF or the presence of defective VWF results in a secondary decrease of FVIII activity and a bleeding tendency known as von Willebrand disease. 7 Two major binding regions for VWF in the FVIII molecule have been reported, one within amino acid residues 1670 to 1689 in the amino-terminal region of LCh 10,11 and the other within residues 2303 to 2332 in the carboxy-terminal region of the C2 domain. 12,13 A3 fragments, composed of amino-terminal residues from 1672 to 1794, and LCh fragments lacking the amino-terminal acidic region of the C2 domain appear to have reduced affinities for VWF, indicating that both regions are essential for the high-affinity binding of VWF to FVIII. 14 The heavy chain is not directly involved in VWF interaction with FVIII. 15 Recently, Jacquemin et al reported that an anti-C1 monoclonal antibody (MoAb) inhibited FVIII binding to VWF, suggesting a possible role of C1 sequences in FVIII and VWF association. 16 Activation and inactivation of FVIII is regulated by VWF, and this regulatory mechanism appears to be essential for the maintenance of normal circulating levels of FVIII activity. Upon activation by thrombin, FVIII dissociates from VWF an...

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Summary. Factor VIII (FVIII) inhibitor alloantibodies react with combinations of the A2, C2 and A3-C1 domains of the FVIII molecule. Some inhibitors block binding of FVIII to both von Willebrand factor (VWF) and phospholipid, and recognize a C2 domain epitope which overlaps both binding sites. In order to determine the essential binding regions for alloantibodies inhibitory for FVIII activity, we have performed inhibitor neutralization assays and competitive inhibition assays using 10 overlapping synthetic peptides spanning the carboxy-terminal region of the C2 domain (residues 2288±2332). We found one peptide (2315±2330, L9) which neutralized the anti-FVIII activity of four out of ®ve different C2 alloantibodies by 50%, 39%, 47% and 57%, respectively. Neutralization of these alloantibodies by recombinant C2 domain (residues 2173±2332) was 68%, 50%, 59%, 86% and >95%, respectively. The inhibitor which was not neutralized by L9 peptide and reacted by immunoblotting with peptide 2218±2307, did not prevent binding of FVIII to VWF and only partially inhibited binding of FVIII to phosphatidylserine. Mutants of the L9 peptide were prepared in which each residue from 2315±2330 was sequentially substituted by glycine. Inhibitor neutralization experiments using these peptides demonstrated that Arg 2320 and Cys 2326or Glu 2327 are important for the effect of L9 peptide, since their substitution by glycine reduced its neutralizing effect by 60% to >90%, suggesting that they are crucial for formation of the one of the C2 inhibitor epitopes.