Functional Analysis of Oligoadenylate Synthetase in the Emu (Dromaius novaehollandiae).

2'-5'-oligoadenylate synthetase (OAS) is one of the proteins that act as a defense mechanism against foreign RNA in cells. OAS has two functions: an antiviral effect against a wide range of virus species via the OAS/RNase L pathway with synthesized oligoadenylates and inhibition of viral replication specific to viruses of the genus Flavivirus, which is independent of enzymatic activity. Several birds have been reported to possess only one type of OAS family member, OASL, which has both enzymatic activity and inhibitory effects on flaviviral replication. However, the ostrich has two types of OASs, OAS1 and OASL, which show different functions-enzymatic and anti-flaviviral activities, respectively. In this study, emu OASs were cloned to investigate their sequence and function and elucidate the role of OASs in emus. The cloning results showed that emus had OAS1 and OASL, suggesting that emu OASs were more closely related to ostrich than to other birds. Functional investigations showed that emu OAS1 and OASL had enzymatic and anti-flaviviral activities, respectively, similar to those of the ostrich. Emus and ostriches are evolutionarily different from most birds and may be more closely related to mammalian OAS diversity.

A Highly Conserved Region in BRCA2 Suppresses the RAD51-Interaction Activity of BRC Repeats.

Mammary tumors are the most prevalent type of tumors in female dogs. Breast cancer 2, early onset (BRCA2) malignant mutations are associated with tumorigenesis in humans and dogs. BRCA2 plays a pivotal role in homologous recombination repair by recruiting RAD51 recombinase to DNA damage sites to maintain genome stability. To recruit RAD51, BRCA2 must interact with RAD51 via BRC repeats, but the regulation of this interaction has been unclear. In this study, we focused on a highly conserved region (HCR) near BRC repeats. Using co-immunoprecipitation and mammalian two-hybrid assay, we found that HCR suppressed the RAD51-interaction activity of BRC repeats and that substitutions of HCR phosphorylation sites affected it. In canine tumor samples, we found ten mutations, including a novel HCR mutation (I1110M) from canine tumor samples. The effect of four HCR mutations, including I1110M, on the RAD51-interaction activity of BRC repeats was tested. One of the HCR mutations found in canine mammary tumors increased the interaction, but the two mutations found in human breast cancers decreased it. This study suggested that the HCR regulated the RAD51-interacting activity of BRC repeats through HCR phosphorylation and that mutations in HCR may be related to tumorigenesis in both dogs and humans.

Canine Mammary Tumor Cell Lines Derived from Metastatic Foci Show Increased RAD51 Expression but Diminished Radioresistance via p21 Inhibition.

Due to the high incidence of mammary tumors in dogs, it is important to elucidate the pathogenesis of these tumors in veterinary medicine. Radiation therapy is often used to treat mammary tumors that target DNA lesions. RAD51 is a key molecule that repairs DNA damage via homologous recombination. We examined the relationship between RAD51 expression and radiosensitivity in mammary tumor cell lines. CHMp and CHMm from the same individual were selected based on the differences in RAD51 expression. The radiosensitivity of both cell lines was examined using MTT and scratch assays; CHMm, which has high RAD51 expression, showed higher sensitivity to radiation than CHMp. However, the nuclear focus of RAD51 during DNA repair was formed normally in CHMp, but not in most of CHMm. Since irradiation resulted in the suppression of cell cycle progression in CHMp, the expression of p21, a cell cycle regulatory factor, was detected in CHMp after 15 Gy irradiation but not in CHMm. These results indicate that functional expression is more important than the quantitative expression of RAD51 in canine mammary tumor cells in response to DNA damage.

BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents.

Breast cancer type 2 susceptibility (BRCA2) protein is crucial for initiating DNA damage repair after chemotherapy with DNA interstrand crosslinking agents or X-ray irradiation, which induces DNA double-strand breaks. BRCA2 contains a C-terminal RAD51-binding domain (CTRBD) that interacts with RAD51 oligomer-containing nucleofilaments. In this study, we investigated CTRBD expression in cells exposed to X-ray irradiation and mitomycin C treatment. Surprisingly, BRCA2 CTRBD expression in HeLa cells increased their resistance to X-ray irradiation and mitomycin C. Under endogenous BRCA2 depletion using shRNA, the sensitivities of the BRCA2-depleted cells with and without the CTRBD did not significantly differ. Thus, the resistance to X-ray irradiation conferred by an exogenous CTRBD required endogenous BRCA2 expression. BRCA2 CTRBD-expressing cells demonstrated effective RAD51 foci formation and increased homologous recombination efficiency, but not nonhomologous end-joining efficiency. To the best of our knowledge, our study is the first to report the ability of the BRCA2 functional domain to confer resistance to X-ray irradiation and mitomycin C treatment by increased homologous recombination efficiency. Thus, this peptide may be useful for protecting cells against X-ray irradiation or chemotherapeutic agents.

In vitro anticancer effects of alpelisib against PIK3CA­mutated canine hemangiosarcoma cell lines.

Hemangiosarcoma (HSA) is a malignant neoplasm that occurs in humans and canines with a poor prognosis owing to metastatic spread, despite effective treatment. The frequency of spontaneous HSA development is higher in canines than in humans. Therefore, canine HSA is a useful model of intractable human disease, which requires early detection and an effective therapeutic strategy. A high frequency of the p110α phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit alpha (PIK3CA) mutations is detected in a comprehensive genome­wide analysis of canine cases of HSA. The present cloned the full­length cDNA of canine PIK3CA and identified a mutation in codon 1047 from canine cases of HSA and cell lines that were established from these. The enforced expression of the 1047th histidine residue (H1047)R or L mutants of canine PIK3CA in HeLa cells enhanced epidermal growth factor receptor (EGFR) signaling via Akt phosphorylation. PIK3CA mutant canine HSA cell lines exhibited the hyperphosphorylation of Akt upon EGF stimulation as well. Alpelisib, a molecular targeted drug against PIK3CA activating mutations, exerted a significant antitumor effect in canine PIK3CA­mutated HSA cell lines. By contrast, it had no significant effect on canine mammary gland tumor cell lines harboring PIK3CA mutations. On the whole, the findings of the present study suggest that alpelisib may be highly effective against PIK3CA mutations that occur frequently in canine HSA.

Identification of the core motif of the BRCA2 C-terminal RAD51-binding domain by comparing canine and human BRCA2.

Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.

Reduced translation efficiency due to novel splicing variants in 5' untranslated region and identification of novel cis-regulatory elements in canine and human BRCA2.

BACKGROUND: Breast cancer 2, early onset (BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination. RESULTS: In this study, the mechanisms that regulate the expression of BRCA2 were proposed. Novel splicing variants were identified in the 5' untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5' UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1. CONCLUSIONS: This study demonstrates that BRCA2 expression level is regulated via 5' UTR splicing variants and that the BRCA2 intron 1 region harbors cis-regulatory elements.

The response of adipose tissues to Mycoplasma pulmonis and Sendai virus infection in C57BL/6 and DBA/2 mice.

Adipose tissues in mammals are categorized into white and brown adipose tissues in which cellular morphology, cell functions, and tissue distribution are different. White adipose tissue (WAT) plays a major role in energy reservation, while brown adipose tissue (BAT) mainly relates to the thermoregulation of the body. One interesting function of adipose tissue is the response to the infection, especially the pathogens that cause pneumonia. We have previously reported that DBA/2 (D2) mice are susceptible to pathogens causing pneumonia, Mycoplasma (M.) pulmonis and Sendai virus (SeV), whereas C57BL/6 (B6) mice are resistant to them. Furthermore, morphological alteration of mediastinal fat tissue (MFT) was seen after infection of M. pulmonis in D2 mice but not in B6 mice. In this study, we aimed to exhibit the difference in adipose tissue response in other areas, including interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (ingWAT), and perigonadal WAT (perigoWAT) between resistant strain, B6 and susceptible strain, D2 after challenging them with M. pulmonis and SeV. Compared with B6 mice, D2 mice showed an increase in fat-associated lymphoid cluster in MFT, an increase in BAT in both iBAT and ingWAT after M. pulmonis and SeV infection. The results of this study indicate that pneumonia caused by M. pulmonis and SeV infection induces browning of adipocyte, suggesting that BAT plays a role in pathogen infection and inflammation.

The number of glutamines in the N-terminal of the canine androgen receptor affects signalling intensities.

Most male dogs are castrated at young ages, making them easy to rear following androgen deprivation. Although the incidence of canine prostate cancer is low, several patients have resistance to androgen therapy and poor clinical prognosis. These outcomes are similar to those of end-stage human androgen-independent prostate cancer. The androgen receptor (AR) of canines has two polyglutamine (polyQ) sequences (Q × 10 and Q × 23) at its N-terminal. The length of polyQ may be a risk factor for the development of prostate cancer in dogs; however, there is no evidence to support this. Hence, we artificially created polyQ deletion mutants of canine AR and evaluated their effects on AR signalling. The deletions of Q × 10 and Q × 23 were associated with significant reductions in AR signalling intensities. The Q × 10 mutants, which increase or decrease Q sequentially, also altered AR signalling. Furthermore, the Q × 10 deletion mutant, compared with the Q × 10 control, altered the intensities of the binding of polyQ to the C-terminal of AR, which contains a ligand-binding domain; this was not observed with the Q × 9, 11, and 12 variants. The number of glutamines in the N-terminals of canine ARs may influence AR signalling intensities and contribute to the risk of prostate cancer in dogs.

Novel canine isocitrate dehydrogenase 1 mutation Y208C attenuates dimerization ability.

Isocitrate dehydrogenase 1 (IDH1) mutations are common in gliomas, acute myeloid leukemia, and chondrosarcoma. The mutation 'hotspot' is a single arginine residue, R132. The R132H mutant of IDH1 produces the 2-hydroxyglutarate (2-HG) carcinogen from α-ketoglutarate (α-KG). The reduction of α-KG induces the accumulation of hypoxia-inducible factor-1α subunit (HIF-1α) in the cytosol, which is a predisposing factor for carcinogenesis. R132H is the most common IDH1 mutation in humans, but mutations at the R132 residue can also occur in tumor tissues of dogs. The current study reported the discovery of a novel Tyr208Cys (Y208C) mutation in canine IDH1 (cIDH1), which was isolated from 2 of 45 canine chondrosarcoma cases. As the genomic DNA isolated from chondrosarcoma tissue was mutated, but that isolated from blood was not, Y208C mutations were considered to be spontaneous somatic mutations. The isocitrate dehydrogenase activity of the Y208C mutant was attenuated compared with that of wild-type (WT) cIDH1, but the attenuation of Y208C was less intense than that of the R132H mutation. The induction of HIF-1α response element activity and cell retention of HIF-1α were not increased by Y208C overexpression. In silico and cell biological analysis of IDH1 dimerization revealed that the Y208C mutation, but not the R132H mutation, attenuated binding activity with WT cIDH1. These data suggested that the attenuation of dimerization by the Y208C mutation may cause tumorigenesis through different mechanisms other than via 2-HG production by the IDH1 R132 mutation.

The canine RAD51 mutation leads to the attenuation of interaction with PALB2.

RAD51 forms a complex with BRCA2 and plays a central role in the DNA damage response pathway that is associated with homologous recombination. The structures of RAD51 and its homologues are highly conserved from prokaryotes to higher eukaryotes. Although a large number of BRCA2 mutations have been reported, there are only a few reports on the mutations of RAD51, which have been shown in humans and dogs. However, several mutations of canine RAD51 were identified from mammary gland tumour tissues in a recent study. Some of these mutations seem to have an influence on the homo-oligomerization or interaction with "Partner and localizer of BRCA2" (PALB2). In this study, we cloned the canine PALB2 homologue and investigated the effect on its interaction with the RAD51 mutants to evaluate the alteration in the function of RAD51 mutants. The A209S and T225S mutants of RAD51 show an attenuation of the interaction between RAD51 and PALB2. These results indicate that the canine RAD51 mutations can potentially alter the homologous recombination pathways in response to DNA damage in dogs.

Null mutation of the endothelin receptor type B gene causes embryonic death in the GK rat.

The Hirschsprung disease (HSCR) is an inherited disease that is controlled by multiple genes and has a complicated genetic mechanism. HSCR patients suffer from various extents of constipation due to dysplasia of the enteric nervous system (ENS), which can be so severe as to cause complete intestinal obstruction. Many genes have been identified as playing causative roles in ENS dysplasia and HSCR, among them the endothelin receptor type B gene (Ednrb) has been identified to play an important role. Mutation of Ednrb causes a series of symptoms that include deafness, pigmentary abnormalities, and aganglionosis. In our previous studies of three rat models carrying the same spotting lethal (sl) mutation on Ednrb, the haplotype of a region on chromosome (Chr) 2 was found to be responsible for the differing severities of the HSCR-like symptoms. To confirm that the haplotype of the responsible region on Chr 2 modifies the severity of aganglionosis caused by Ednrb mutation and to recreate a rat model with severe symptoms, we selected the GK inbred strain, whose haplotype in the responsible region on Chr 2 resembles that of the rat strain in which severe symptoms accompany the Ednrbsl mutation. An Ednrb mutation was introduced into the GK rat by crossing with F344-Ednrbsl and by genome editing. The null mutation of Ednrb was found to cause embryonic death in F2 progeny possessing the GK haplotype in the responsible region on Chr 2. The results of this study are unexpected, and they provide new clues and animal models that promise to contribute to studies on the genetic regulatory network in the development of ENS and on embryogenesis.

Profiling of cellular immune responses to Mycoplasma pulmonis infection in C57BL/6 and DBA/2 mice.

Mycoplasma infections cause respiratory tract damages and atypical pneumonia, resulting in serious problems in humans and animals worldwide. It is well known that laboratory inbred mouse strains show various susceptibility to Mycoplasma pulmonis (M. pulmonis) infection, which causes murine respiratory mycoplasmosis. In this study, we aimed to demonstrate the difference in cellular immune responses between resistant strain, C57BL/6NCrSlc (B6) and susceptible strain, DBA/2CrSlc (D2) after challenging M. pulmonis infection. D2 mice showed higher amount of bacterial proliferation in lung, higher pulmonary infiltration of immune cells such as neutrophils, macrophages, and lymphocytes, and higher levels of interleukin (IL)-1ß, IL-6, IL-17A, and tumor necrosis factor-α in bronchoalveolar lavage fluid than did B6 mice. The results of this study suggest that D2 mice are more susceptible than B6 mice to M. pulmonis infection due to a hyper-immune inflammatory response.

Analysis for genetic loci controlling protoscolex development in the Echinococcus multilocularis infection using congenic mice.

The resistance/susceptibility to Echinococcus multilocularis infection in mice is genetically controlled. However, genetic factors responsible for these differences remain unknown. Our previous study in genetic linkage analysis has revealed that there is a significant quantitative trait locus (QTL) for the establishment of cyst (Emcys1), and a highly significant QTL for the development of protoscolex of E. multilocularis larvae (Empsc1), on mouse chromosomes 6 and 1, respectively. The current study aimed to confirm these QTLs and narrow down the critical genetic region that controls resistance/susceptibility to E. multilocularis infection by establishing congenic and subcongenic lines from C57BL/6 (B6) and DBA/2 (D2) mice. For protoscolex development phenotype, two congenic lines, B6.D2-Empsc1 and D2.B6-Empsc1 were developed, where responsible QTL, Empsc1 was introgressed from D2 into B6 background and vice versa. For cyst establishment phenotype, two congenic lines, B6.D2-Emcys1 and D2.B6-Emcys1 were developed, where responsible QTL, Emcys1 was introgressed from D2 into B6 background and vice versa. Because there was no significant difference in cyst establishment between B6.D2-Emcys1 and D2.B6-Emcys1 mice after challenge with E. multilocularis, it is suggested that the Emcys1 does not solely control the cyst establishment in mouse liver. However, infection experiments with B6.D2-Empsc1 and D2.B6-Empsc1 mice showed a significant difference in protoscolex development in the cyst. It confirms that the Empsc1 controls phenotype of the protoscolex development in the cyst. Subsequently, two subcongenic lines, B6.D2-Empsc1.1 and B6.D2-Empsc1.2 from B6.D2-Emcys1 and one subcongenic line, D2.B6-Empsc1.1 from D2.B6-Empsc1 were developed to narrow down the critical region responsible for protoscolex development. From the results of infection experiments with E. multilocularis in these subcongenic mice, it is concluded that a gene responsible for protoscolex development is located between D1Mit290 (68.1 cM) and D1Mit511 (97.3 cM).

Endogenous Leu332Gln mutation in p53 disrupts the tetramerization ability in a canine mammary gland tumor cell line.

Mutations in the p53 gene are associated with more than half of all human cancers. These mutations often cause a disruption of the tumor-suppressor function of p53 and induce genomic instabilities. Wild­type p53 requires tetramerization to function as an initiator of cell cycle arrest and apoptosis. Although alterations in p53 tetramerization caused by mutation have been well studied, there are few cell lines containing an endogenous mutation in the tetramerization domain of p53. Here, we report the discovery of a canine mammary gland tumor cell line CTB­m2, which contains the Leu332Gln (L332Q) mutation corresponding to Leu344 in the tetramerization domain of human p53. Although CTB­m2 cells are genetically heterozygous for the Leu332Gln mutation, the mutant mRNA was almost exclusively expressed. CTB­m2 cells showed enhanced cell proliferation compared to wild­type p53-expressing CTB­m cells of the same lineage. A p53 tetramerization reporter assay showed that the ability of the p53 mutant to form tetramers was significantly lower than that of wild­type p53. An immunoblot analysis of cross-linked p53 oligomerized forms demonstrated that the L332Q mutant lacked the ability to form tetramers but retained the ability to form dimers. These data suggest that the p53 mutant cell line CTB­m2 could be a useful tool for analyzing the precise tetramerization mechanisms of p53 and verifying the effects of therapeutic agents against tumors expressing p53 mutants that lack the ability to tetramerize.

Functional analysis of duck, goose, and ostrich 2'-5'-oligoadenylate synthetase.

Up-to-date the flavivirus infection in avian taxa is not clearly defined. Several reports have demonstrated that many viruses belonging to Flaviviridae may cause diseases in poultry species; however, the susceptibility of other avian species is variable and still unclear. In human and mice, the 2'-5'-oligoadenylate synthetase (OAS) proteins are associated with resistance to the flavivirus infection as well as other virus infections. However, the avian OAS proteins are rarely studied. In our previous studies, we confirmed that the chicken OAS-like protein (chOASL) expressed OAS-enzymatic activity (the classical OAS/RNase L-dependent pathway) as well as the anti-flavivirus activity (the putative OAS/RNase L-independent pathway). Therefore, the current study aimed at functional analysis of avian OAS proteins from duck, goose, and ostrich. The duOASL, goOASL, and osOAS1 proteins expressed enzymatic activity as well as chOASL, whereas osOASL expressed little enzymatic activity. On the other hand, duOASL, goOASL, and osOASL possessed significant antiviral activity against West Nile virus (WNV)-replicon replication as well as chOASL, whereas osOAS1 did not. In addition, similar to chOASL, their antiviral activity was independent of RNase L activation. These results suggest that OASL is the only OAS protein in the duck and goose as well as chicken and possesses both OAS-enzymatic and anti-flavivirus activities, whereas the ostrich possesses both OAS1 and OASL proteins with sharing the functional activities, OAS-enzymatic and anti-flavivirus activities, respectively. It is of interest that the ostrich undergoes differential process in OAS gene evolution from other poultries and thus possesses different molecular mechanism in antiviral activity.

Verification of genetic loci responsible for the resistance/susceptibility to the Sendai virus infection using congenic mice.

Sendai virus (SeV) is one of the most important pathogens in the specific-pathogen free rodents. It is known that there are some inbred mouse strains susceptible or resistant to SeV infection. The C57BL/6 (B6) and DBA/2 (D2) mice are representative of the resistant and susceptible strains, respectively. Previous study with the quantitative trait locus (QTL) analysis identified three QTLs responsible for resistance or susceptibility to SeV infection on different chromosomes and indicated that resistance or susceptibility to SeV infection was almost predicted by genotypes of these three QTLs. In this paper, to verify the above hypothesis, congenic lines were generated as follows; B6-congenic lines carrying one of the D2 alleles of three QTLs and combination of these three QTLs, and D2-congenic lines carrying single or combination of B6 alleles of three QTLs. All these congenic lines were then challenged with SeV infection. D2 congenic lines introgressed single or combination of B6 alleles of QTLs changed to resistance to SeV infection. Especially, a D2 triple-congenic line became resistant as similar level to B6-parental strain. However, B6-congenic lines introgressed single or combination of D2 alleles of QTLs all remained to be resistant to SeV infection. Both IL-6 and TNF-α in broncho-alveolar lavage fluid of D2 triple-congenic line were decreased to the similar level of B6 mice, suggesting that this is a part of factors that D2 triple-congenic line became resistant to the similar level of B6 mice. Data obtained from these congenic mice verified that three QTLs identified previously were indeed responsible for the resistance/susceptibility to SeV infection in B6 and D2 mice.

R132 mutations in canine isocitrate dehydrogenase 1 (IDH1) lead to functional changes.

Glioma is the second most common intracranial neoplasia in dogs, but the pathogenic mechanisms remain unclear. In humans, isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas. Although almost all human IDH1 mutations have been identified as involving the Arg132 codon, few studies have reported structural, functional, and mutational information for canine IDH1. Therefore, in this study, we cloned the canine IDH1 homologue and used PCR mutagenesis to substitute the wildtype (WT) Arg132 with His (R132H) or Ser (R132S). WT and mutated IDH1 were overexpressed in HeLa cells, and their presence was confirmed by immunoblotting and immunocytochemistry using mutation-specific antibodies. The IDH1 activity between WT, R132H, and R132S transfectants was compared by measuring the production of NADH and NADPH. NADPH production in R132H and R132S transfectants was lower than that in WT, but NADH levels were not significantly different. Finally, we detected increased expression of hypoxia inducible factor 1 alpha (HIF-1α) in the R132H and R132S transfectants. These results indicated that the canine IDH1 Arg132 mutation has the potential to induce carcinogenesis in canine somatic cells.

Comparison of the antiviral potential among soluble forms of herpes simplex virus type-2 glycoprotein D receptors, herpes virus entry mediator A, nectin-1 and nectin-2, in transgenic mice.

Herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2 are cellular receptors of glycoprotein D (gD) of herpes simplex virus type-2 (HSV-2). It has been shown that soluble forms of HSV gD receptors have the antiviral potential in cultured cells and transgenic mice. Here, to compare antiviral potential of soluble forms of HVEM, nectin-1 and nectin-2 against HSV-2 infections in vivo, transgenic mice expressing fusion proteins consisting of the entire ectodomain of HVEM, nectin-1 or nectin-2 and the Fc portion of human IgG (HVEMIg, nectin-1Ig and nectin-2Ig, respectively) were intraperitoneally infected with HSV-2. In the infection with 3 MLD50 (50 % mouse lethal dose), effective resistance was not observed in transgenic mice expressing nectin-2Ig. In a transgenic mouse line with high expression of nectin-1Ig, significant protection from the infection with 30 and 300 MLD50 was observed (survival rate of 100 and 71 %, respectively). On the other hand, transgenic mice expressing HVEMIg showed a complete resistance to the lethal infection even with 300 MLD50 (survival rate of 100 %). These results demonstrated that HVEMIg could exert effective antiviral activities against HSV-2 infections in vivo as compared with other soluble forms of HSV gD receptors.

Cell-cycle arrest in mature adipocytes impairs BAT development but not WAT browning, and reduces adaptive thermogenesis in mice.

We previously reported brown adipocytes can proliferate even after differentiation. To test the involvement of mature adipocyte proliferation in cell number control in fat tissue, we generated transgenic (Tg) mice over-expressing cell-cycle inhibitory protein p27 specifically in adipocytes, using the aP2 promoter. While there was no apparent difference in white adipose tissue (WAT) between wild-type (WT) and Tg mice, the amount of brown adipose tissue (BAT) was much smaller in Tg mice. Although BAT showed a normal cellular morphology, Tg mice had lower content of uncoupling protein 1 (UCP1) as a whole, and attenuated cold exposure- or ß3-adrenergic receptor (AR) agonist-induced thermogenesis, with a decrease in the number of mature brown adipocytes expressing proliferation markers. An agonist for the ß3-AR failed to increase the number of proliferating brown adipocytes, UCP1 content in BAT, and oxygen consumption in Tg mice, although the induction and the function of beige adipocytes in inguinal WAT from Tg mice were similar to WT mice. These results show that brown adipocyte proliferation significantly contributes to BAT development and adaptive thermogenesis in mice, but not to induction of beige adipocytes.