A multi-tasking stomach: functional coexistence of acid-peptic digestion and defensive body inflation in three distantly related vertebrate lineages.

Puffer and porcupine fishes (families Diodontidae and Tetraodontidae, order Tetradontiformes) are known for their extraordinary ability to triple their body size by swallowing and retaining large amounts of seawater in their accommodating stomachs. This inflation mechanism provides a defence to predation; however, it is associated with the secondary loss of the stomach's digestive function. Ingestion of alkaline seawater during inflation would make acidification inefficient (a potential driver for the loss of gastric digestion), paralleled by the loss of acid-peptic genes. We tested the hypothesis of stomach inflation as a driver for the convergent evolution of stomach loss by investigating the gastric phenotype and genotype of four distantly related stomach inflating gnathostomes: sargassum fish, swellshark, bearded goby and the pygmy leatherjacket. Strikingly, unlike in the puffer/porcupine fishes, we found no evidence for the loss of stomach function in sargassum fish, swellshark and bearded goby. Only the pygmy leatherjacket (Monochanthidae, Tetraodontiformes) lacked the gastric phenotype and genotype. In conclusion, ingestion of seawater for inflation, associated with loss of gastric acid secretion, is restricted to the Tetraodontiformes and is not a selective pressure for gastric loss in other reported gastric inflating fishes.

The male-specific factor Sry harbors an oncogenic function.

Sgf29, a component of the SPT-ADA-GCN5 acetyltransferase (SAGA) complex, binds H3K4me2/3 marks and leads to histone H3 acetylation. Previously, we found that downregulation of Sgf29 suppresses c-Myc-mediated malignant transformation. Nonetheless, the upstream regulator of the Sgf29 gene is not yet known. Here, we report that Sry (sex-determining region Y), an HMG (high-mobility group) domain containing transcription factor, directly upregulates Sgf29 gene expression. Sry expression was deregulated in two out of the four tested male rodent hepatocellular carcinoma (rHCC) cell lines. Luciferase reporter and chromatin immunoprecipitation assays indicated that Sry could bind HMG-boxes in the proximal promoter region of the Sgf29 gene. Knockdown of Sry robustly lowered anchorage-independent growth, invasiveness and tumorigenicity of rHCC cells, whereas ectopic expression of Sry conferred more malignant properties. Thus, these data show that Sry is involved in male-specific malignant conversion of rHCCs via Sgf29 upregulation.

p53 target gene AEN is a nuclear exonuclease required for p53-dependent apoptosis.

DNA degradation is one of the biochemical hallmarks detected in apoptotic cells, and several nucleases have been reported to function cooperatively in this process. It has also been suggested that different sets of nucleases are activated by different stimuli, and induce distinct patterns of DNA degradation. Here we report that apoptosis-enhancing nuclease (AEN) is a novel direct target gene of p53. AEN is induced by p53 with various DNA damage, and its expression is regulated by the phosphorylation status of p53. We demonstrate that AEN is a typical exonuclease with conserved exonuclease domains Exo I-III, and it targets both single- and double-stranded DNA and RNA. AEN induces apoptosis by itself, and the conserved domains are essential for both AEN nuclease activity and its apoptosis-inducing ability. AEN possesses nuclear and nucleolar localization signals, and it translocates from the nucleolus to nucleoplasm upon apoptosis induction. We also show the dislocation of nucleophosmin in conjunction with the translocation of AEN to the nucleoplasm, indicating the ability of AEN in nucleolus disruption. In addition, AEN is shown to be required for efficient DNA fragmentation in p53-dependent apoptosis. These results suggest that AEN is an important downstream mediator of p53 in apoptosis induction.

Phosphorylation of PML is essential for activation of C/EBP epsilon and PU.1 to accelerate granulocytic differentiation.

Promyelocytic leukemia (PML) is a nuclear protein that functions as a regulator of transcription, cell proliferation, apoptosis and myeloid cell differentiation. PML is subjected to post-translational modifications such as sumoylation and phosphorylation. However, the physiological significance of these modifications, especially for myeloid cell differentiation, remains unclear. In this report, we found that four serine residues in the PML C-terminal region are highly phosphorylated in a myeloid cell line. Wild-type PML accelerated G-CSF-induced granulocytic differentiation, but a phosphorylation-deficient PML mutant failed. PML interacted with C/EBP epsilon, a transcription factor essential for granulopoiesis, activated C/EBP epsilon-mediated transcription in concert with p300 and accelerated C/EBP epsilon-induced granulocytic differentiation. Phosphorylation of PML was required for stimulating C/EBP epsilon-dependent transcription and accelerating C/EBP epsilon-induced granulocytic differentiation. We also found that PML phosphorylation was required for stimulation of PU.1-dependent transcription and acceleration of PU.1-induced granulocytic differentiation. These results suggest that phosphorylation plays essential roles in the regulation of PML to accelerate granulocytic differentiation through multiple pathways.

Pdx-1-independent differentiation of mouse embryonic stem cells into insulin-expressing cells.

To investigate whether insulin-producing cells obtained from ES cells via the nestin-positive cell-mediated method are of the pancreatic lineage, we established a pdx-1 knockout ES cell line and analyzed its differentiation into insulin-producing cells. As a result, pdx-1 knockout ES cell expressed insulin 2 gene at the final differentiated cells. Thus, our study demonstrated that pdx-1 is not essential for insulin gene expression, at least in cells differentiated from this population of nestin-expression enriched ES cells, and suggested that the insulin-producing cells derived from ES cells may be different from the pancreatic beta cells in terms of their lineage.

Deregulated expression of a novel component of TFTC/STAGA histone acetyltransferase complexes, rat SGF29, in hepatocellular carcinoma: possible implication for the oncogenic potential of c-Myc.

c-Myc N-terminal conserved domains, MbI and MbII, are essential for c-Myc-mediated transformation and transactivation. These domains recruit the STAGA (SPT3-TAF9-GCN5-acetyltransferase) coactivator complex, but not TFTC (TATA-binding protein-free TAF-containing) to the target gene promoter. Although components of this complex are well conserved between yeast and mammals, four mammalian orthologs of yeast SPT8, SPT20, SGF11 and SGF29 remain to be identified. Here, we isolated a rat ortholog of yeast SGF29, a component of yeast SAGA (SPT-ADA-GCN5-acetyltransferase) complex. Both rat (r) SGF29 and c-myc mRNAs were overexpressed in five out of the eight tested rodent tumor cells. rSGF29 directly interacted with rADA3 and co-immunoprecipitated with two other TFTC/STAGA components, rGCN5 and rSPT3. rSGF29 was recruited to the c-Myc target gene promoters together with c-Myc, and it activated c-Myc target gene expressions. Downregulation of rSGF29 suppressed the expression of c-Myc target genes and inhibited anchorage-independent growth and tumorigenicity and lung metastasis of rat hepatoma K2 cells when injected into nude mice. These results show that rSGF29 is a novel component of TFTC/STAGA complexes and could be involved in the c-Myc-mediated malignant transformation.

Stimulation of cAMP signalling allows isolation of clonal pancreatic precursor cells from adult mouse pancreas.

AIMS/HYPOTHESIS: Duct cells of the pancreas are thought to include latent progenitors of islet endocrine cells that can be induced to differentiate by appropriate morphogens. Here we developed a method for isolating pancreatic ductal epithelial cells from adult mice that overcomes the shortcomings of previous methods. MATERIALS AND METHODS: Pancreatic ductal cells were grown in serum-free DMEM/F12 medium in the presence of cholera toxin or 8-bromo-cyclic adenosine monophosphate, which is known to be an intracellular cAMP generator. Single cell cloning was performed by limiting dilution in serum-free medium. RESULTS: The isolated clonal cells expressed high levels of cytokeratin and Ipf1 (formerly known as Pdx-1). Adenovirus-mediated expression of ngn3 (also known as Neurog3) and Ptf1a in these cells induced expression of insulin and somatostatin, and of carboxypeptidase A, respectively. Furthermore, albumin production was induced by dexamethasone or by long-term culture in serum-containing medium. CONCLUSIONS/INTERPRETATION: Stimulation of the cAMP-dependent signalling allowed us to isolate clonal pancreatic ductal cells from adult mice. These cells are able to partially differentiate into endocrine cells, exocrine cells and hepatocyte-like cells and are therefore considered to have the characteristics of endodermal progenitor cells.

Protective role for cytosolic phospholipase A2alpha in autoimmune diabetes of mice.

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) plays an important role in arachidonate pathway. To investigate the contribution of cPLA(2)alpha to autoimmune diabetes, we established non-obese diabetic (NOD) mouse, an excellent model for human type 1 diabetes, deficient in cPLA(2)alpha. These mice showed severe insulitis and a higher incidence of diabetes. In their macrophages, decreased prostaglandin E(2) (PGE(2)) induced by cPLA(2)alpha deficiency, and the increase in production of tumor necrosis factor (TNF)-alpha were observed. These results suggested that cPLA(2)alpha plays a protective role in progression of insulitis and development of autoimmune diabetes by suppression of TNF-alpha production from macrophages.

Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32.

Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrk-mediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32(74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.

Development of autoimmune diabetes in glutamic acid decarboxylase 65 (GAD65) knockout NOD mice.

AIMS/HYPOTHESIS: Type 1 diabetes mellitus, a T-cell-mediated autoimmune disease, results from the selective destruction of insulin-producing pancreatic beta cells. Autoantibodies against beta-cell components are used clinically as sensitive markers of this disease; however, their physiological role has not been clear. To investigate the role of glutamic acid decarboxylase 65 (GAD65) in the development of the Type 1 diabetes of non-obese diabetic (NOD) mice, we analysed and characterised NOD mice with targeted disruption of the GAD65 gene. METHODS: GAD65-deficient mice were previously established. After backcrossing the knockout mutation onto the NOD genetic background for up to eight generations, female littermates of the three resulting genotypes were produced by intercrossing: GAD65 +/+ (n=23), GAD65 +/- (n=62), and GAD65 -/- (n=31). RESULTS: The cumulative incidence of autoimmune diabetes showed no significant difference among the three groups in longitudinal studies using the Kaplan-Meier method. Islet morphology showed that the progression of islet infiltration did not differ significantly between the three groups. CONCLUSION/INTERPRETATION: The cumulative incidence of autoimmune diabetes was not influenced by the GAD65 deficiency. These data suggest that GAD65 is not a major regulatory target of beta-cell autoimmunity in NOD mice.

beta-cell neogenesis induced by adenovirus-mediated gene delivery of transcription factor pdx-1 into mouse pancreas.

beta-cell neogenesis is expected to provide a new therapy for diabetes. Numerous studies have demonstrated that transcriptional regulation involving pdx-1 is essential for endocrine neogenesis in vivo and in vitro. Therefore, it is possible that ectopic expression of pdx-1 in the pancreas could induce endocrine neogenesis. To test this possibility, we performed safe and efficient gene delivery of the pdx-1 gene into the mouse pancreas through the common bile duct using adenoviral vectors, and examined the effects of the ectopic expression of pdx-1. Here we show that adenovirus-mediated expression of pdx-1 can activate the endogenous pdx-1 gene, leading to beta-cell neogenesis and ductal proliferation. This technique is similar to the endoscopic retrograde cholangiopancreatography, which has been already established as a safe procedure for humans. Thus, beta-cell neogenesis induced by adenovirus-mediated expression of pdx-1 provides a novel strategy for gene therapy for a cure for diabetes mellitus.

M6a acts as a nerve growth factor-gated Ca(2+) channel in neuronal differentiation.

To elucidate the function of M6a, which is a neuron-specific membrane glycoprotein of the brain and possesses putative phosphorylation sites for protein kinase C (PKC), we established rat M6a cDNA expression vector-transfected PC12 cells. These transfectants exhibited high susceptibilities to nerve growth factor (NGF) for neuronal differentiation. Interestingly, we found that Ca(2+) influx in these transfectants was significantly augmented by the treatment of NGF, but not epidermal growth factor (EGF), which stimulates PC12 cell growth. NGF-dependent augmentation of Ca(2+) influx was detected within 3h and severely inhibited by EGTA- and PKC-specific inhibitors. Anti-M6 antibody suppressed both NGF-triggered Ca(2+) influx and neuronal differentiation. These results support the idea that M6a implicates in neuronal differentiation as a novel Ca(2+) channel gated selectively by phosphorylation with PKC in the downstream of NGF signaling pathway.

Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP).

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been conserved remarkably during evolution and is widely expressed in the mammalian brain. In Drosophila, mutation of the PACAP homologue results in behavioral defects, including impaired olfaction-associated learning and changes in ethanol sensitivity. Here, we report the generation of mice lacking the PACAP gene (PACAP(-/-)). PACAP(-/-) mice were born in the expected Mendelian ratios but had a high early-mortality rate. The surviving adult PACAP(-/-) mice displayed remarkable behavioral changes; they exhibited hyperactive and explosive jumping behaviors in an open field, increased exploratory behavior, and less anxiety in the elevated plus maze, emergence, and novel-object tests. Analysis of PACAP(-/-) mice brains revealed that the serotonin metabolite 5-hydroxyindoleacetic acid was slightly decreased in the cortex and striatum compared with wild-type mice. The present study provides evidence that PACAP plays a previously uncharacterized role in the regulation of psychomotor behaviors.

Loss of hippocampal CA3 pyramidal neurons in mice lacking STAM1.

STAM1, a member of the STAM (signal transducing adapter molecule) family, has a unique structure containing a Src homology 3 domain and ITAM (immunoreceptor tyrosine-based activation motif). STAM1 was previously shown to be associated with the Jak2 and Jak3 tyrosine kinases and to be involved in the regulation of intracellular signal transduction mediated by interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Here we generated mice lacking STAM1 by using homologous recombination with embryonic stem cells. STAM1(-/-) mice were morphologically indistinguishable from their littermates at birth. However, growth retardation in the third week after birth was observed for the STAM1(-/-) mice. Unexpectedly, despite the absence of STAM1, hematopoietic cells, including T- and B-lymphocyte and other hematopoietic cell populations, developed normally and responded well to several cytokines, including IL-2 and GM-CSF. However, histological analyses revealed the disappearance of hippocampal CA3 pyramidal neurons in STAM1(-/-) mice. Furthermore, we observed that primary hippocampal neurons derived from STAM1(-/-) mice are vulnerable to cell death induced by excitotoxic amino acids or an NO donor. These data suggest that STAM1 is dispensable for cytokine-mediated signaling in lymphocytes but may be involved in the survival of hippocampal CA3 pyramidal neurons.

Suppression of T(h)1 cell activation and prevention of autoimmune diabetes in NOD mice by local expression of viral IL-10.

Insulin-dependent diabetes mellitus in the NOD mouse model is caused by the T cell-mediated autoimmune destruction of pancreatic beta cells. Viral IL-10 (vIL-10), encoded in the Epstein-Barr virus genome, shares many of the anti-inflammatory properties of cellular IL-10, but lacks its immunostimulatory properties. In the present study, we generated transgenic (Tg) NOD mice in which vIL-10 was produced exclusively in pancreatic islets and investigated the effect of vIL-10 on the development of diabetes. The accumulation of lymphocytes around islets was more prominent, but the invasive insulitis decreased in the vIL-10 Tg mice. The incidence of diabetes was markedly reduced in the vIL-10 Tg mice, in clear contrast to the accelerated diabetes seen in the murine IL-10 Tg NOD mice. IL-12p40 and IFN-gamma mRNA levels were decreased in pancreata of the vIL-10 Tg mice, although CD4 mRNA level was markedly increased. These results suggest that locally produced vIL-10 induced leukocyte migration, but inhibited the activation of T(h)1, probably through suppressing the production of IL-12. They indicate that vIL-10 may well be superior to cellular IL-10 in the treatment of autoimmune diabetes. The vIL-10 Tg NOD mice should provide a useful tool for understanding the differential action of vIL-10 versus cellular IL-10.

IL-12 plays a pathologic role at the inflammatory loci in the development of diabetes in NOD mice.

Accumulating evidence suggests that CD4(+)T helper type 1 (Th1) cells play a major role in the development of insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse model. Interleukin (IL)-12 is a potent immunoregulatory molecule that is a key determinant of T-cell differentiation into Th1 cells, and has been implicated in the development of IDDM. To investigate the role of IL-12 that is locally produced by islet-infiltrating cells in the development of IDDM, we generated transgenic NOD mice in which the IL-12 p40 homodimer, a natural antagonist of IL-12, was produced exclusively in islets without affecting the levels of IL-12 p40 in the systemic circulation. We found that the incidence of diabetes was significantly reduced in these transgenic mice. These results clearly demonstrate that IL-12 locally produced by islet-infiltrating cells plays a critical role in the development of IDDM.

Structure of the mouse glutamate decarboxylase 65 gene and its promoter: preferential expression of its promoter in the GABAergic neurons of transgenic mice.

GABA is synthesized by glutamate decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5'-flanking region indicated the presence of potential neuron-specific cis-regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high beta-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression.

Protective immune responses induced by a non-pathogenic simian/human immunodeficiency virus (SHIV) against a challenge of a pathogenic SHIV in monkeys.

A simian/human immunodeficiency virus (SHIV)-NM3n containing the human nef, but not the monkey nef, and vpr genes of SIV was inoculated into two cynomolgus monkeys, resulting in systemic infection with a minimum level of transient virus load. In order to study the nature of immune responses associated with the prevention of a pathogenic SHIV, the SHIV-NM3n-inoculated monkeys and three naive monkeys were intravenously challenged with a pathogenic SHIV containing the envelope gene of HIV-1 89.6. After the heterologous virus challenge, all of the SHIV-NM3n-inoculated animals completely avoided the loss of CD4+ T lymphocytes in PBMC as well as lymphoid tissues compared to pathogenic SHIV-injected control animals. The inhibition of CD4+ cell depletion was associated with maintaining the proliferative response of helper T-cells against SIV p27 in the previously nonpathogenic virus-inoculated animals following the pathogenic virus challenge. Furthermore, the decline of CD28+ cells, the increase in CD95+ cells, and the enhancement of in vitro apoptosis in PBMC were inhibited in the non-pathogenic virus-inoculated animals. These results suggest that nonpathogenic SHIV-NM3n infection induces the protection of monkeys from heterologous pathogenic viruses that may be associated with blocking the change in immune responses and the cell loss induced by a pathogenic virus.

Upgrading of flow cytometric analysis for absolute counts, cytokines and other antigenic molecules of cynomolgus monkeys (Macaca fascicularis) by using anti-human cross-reactive antibodies.

In order to effectively use cynomolgus monkeys as animal models for human diseases, more than 300 anti-human monoclonal antibodies (mAbs) were studied as to their cross-reaction with various antigens from cynomolgus monkeys (Macaca fascicularis). Two hundred twenty-nine of 339 (67.55%) anti-human mAbs that react with human antigens of CD-defined molecules, chemokine receptors, and T cell receptors were cross-reactive with the monkey antigens. Using the cross-reactive antibodies and the fluorescenced beads for calibration, the procedure for the absolute count of monkey lymphocyte subsets was developed and the mean values for CD4+ and CD8+ lymphocyte subsets in peripheral blood were 718 and 573/mm3, respectively. Moreover, intracellular cytokines, IL-2, IL-4 and IFN gamma, and intracellular apoptosis-related proteins, Bcl-2, FADD and active form of caspase-3 could be detected in peripheral blood mononuclear cells as well as various tissue cells. It is therefore practicable to detail the phenotype of leukocytes, assess the production of intracellular cytokines and enumerate T-lymphocyte subsets by using the cross-reactive human antibodies with respective antigens of cynomolgus monkeys.

A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination.

The success of Cre-mediated conditional gene targeting depends on the specificity of Cre recombinase expression in Cre-transgenic mouse lines. As a tool to evaluate the specificity of Cre expression, we developed a reporter transgenic mouse strain that expresses enhanced green fluorescent protein (EGFP) upon Cre-mediated recombination. We demonstrate that the progeny resulting from a cross between this reporter strain and a transgenic strain expressing Cre in zygotes show ubiquitous EGFP fluorescence. This reporter strain should be useful to monitor the Cre expression directed by various promoters in transgenic mice, including mice in which Cre is expressed transiently during embryogenesis under a developmentally regulated promoter.