Mitsuo Maruyama

PEBP2 alpha B/mouse AML1 consists of multiple isoforms that possess differential transactivation potentials.

A murine transcription factor, PEBP2, is composed of two subunits, alpha and beta. There are two genes in the mouse genome, PEBP2 alpha A and PEBP2 alpha B, which encode the alpha subunit. Two types of the alpha B cDNA clones, alpha B1 and alpha B2, were isolated from mouse fibroblasts and characterized. They were found to represent 3.8- and 7.9-kb transcripts, respectively. The 3.8-kb RNA encodes the previously described alpha B protein referred to as alpha B1, while the 7.9-kb RNA encodes a 387-amino-acid protein, termed alpha B2, which is identical to alpha B1 except that it has an internal deletion of 64 amino acid residues. Both alpha B1 and alpha B2 associate with PEBP2 beta and form a heterodimer. The alpha B2/beta complex binds to the PEBP2 binding site two- to threefold more strongly than the alpha B1/beta complex does. alpha B1 stimulates transcription through the PEBP2 site about 40-fold, while alpha B2 is only about 25 to 45% as active as alpha B1. Transactivation domain is located downstream of the 128-amino-acid runt homology region, referred to as the Runt domain. Mouse chromosome mapping studies revealed that alpha A, alpha B, and beta genes are mapped to chromosomes 17, 16, and 8, respectively. The last two genes are syntenic with the human AML1 on chromosome 21q22 and PEBP2 beta/CBF beta on 16q22 detected at the breakpoints of characteristic chromosome translocations of the two different subtypes of acute myeloid leukemia. These results suggest that previously described chimeric gene products, AML1/MTG8(ETO) and AML1-EAP generated by t(8;21) and t(3;21), respectively, lack the transactivation domain of AML1.

The integrity of the conserved 'WS motif' common to IL-2 and other cytokine receptors is essential for ligand binding and signal transduction.

Recent studies have identified a new family of cytokine receptors, which is primarily characterized by the conservation of periodically interspersed four cysteine residues and the W‐S‐X‐W‐S sequence (‘WS motif’) within the extracellular domain. However, the role of such conserved structures still remains elusive, in particular that of the WS motif. Interleukin‐2 (IL‐2) is known to play a critical role in the clonal expansion of antigen‐stimulated T lymphocytes, and the IL‐2 signal is delivered by one of the receptor components, the IL‐2 receptor beta (IL‐2R beta) chain. The IL‐2R beta chain, unlike the IL‐2R alpha chain, belongs to this receptor family. In the present study, we analyzed the function of the WS motif of IL‐2R beta (Trp194‐Ser195‐Pro196‐Trp197‐Ser198) with the use of site‐directed mutagenesis. Our results indicate the critical role of the two Trp residues in the proper folding of the IL‐2R beta extracellular domain and point to the general functional importance of the WS motif in the new cytokine receptor family.

Subcellular localization of the alpha and beta subunits of the acute myeloid leukemia-linked transcription factor PEBP2/CBF.

Each of the two human genes encoding the alpha and beta subunits of a heterodimeric transcription factor, PEBP2, has been found at the breakpoints of two characteristic chromosome translocations associated with acute myeloid leukemia, suggesting that they are candidate proto-oncogenes. Polyclonal antibodies against the alpha and beta subunits of PEBP2 were raised in rabbits and hamsters. Immunofluorescence labeling of NIH 3T3 cells transfected with PEBP2 alpha and -beta cDNAs revealed that the full-size alpha A1 and alpha B1 proteins, the products of two related but distinct genes, are located in the nucleus, while the beta subunit is localized to the cytoplasm. Deletion analysis demonstrated that there are two regions in alpha A1 responsible for nuclear accumulation of the protein: one mapped in the region between amino acids 221 and 513, and the other mapped in the Runt domain (amino acids 94 to 221) harboring the DNA-binding and the heterodimerizing activities. When the full-size alpha A1 and beta proteins are coexpressed in a single cell, the former is present in the nucleus and the latter still remains in the cytoplasm. However, the N- or C-terminally truncated alpha A1 proteins devoid of the region upstream or downstream of the Runt domain colocalized with the beta protein in the nucleus. In these cases, the beta protein appeared to be translocated into the nucleus passively by binding to alpha A1. The chimeric protein containing the beta protein at the N-terminal region generated as a result of the inversion of chromosome 16 colocalized with alpha A1 to the nucleus more readily than the normal beta protein. The implications of these results in relation to leukemogenesis are discussed.

Zizimin2: a novel, DOCK180-related Cdc42 guanine nucleotide exchange factor expressed predominantly in lymphocytes

A novel superfamily of guanine nucleotide exchange factors for Rho GTPases includes DOCK180 and zizimin1. The zizimin subfamily includes three genes of which only zizimin1 has been cloned. We report here the cloning of zizimin2, identified in a screen for genes enriched in germinal center B cells. Zizimin2 and zizimin1 have similar primary structures and both proteins bound and activated Cdc42 but not the Cdc42‐related proteins TC10 or TCL. Their tissue distributions are distinct, however, with zizimin2 expressed predominantly in lymphocytes and an opposite pattern for zizimin1. Zizimin3 was also analyzed and showed distinct GTPase specificity and tissue distribution.

Clinical effects of probiotic Bifidobacterium longum BB536 on immune function and intestinal microbiota in elderly patients receiving enteral tube feeding.

BACKGROUND Immune system function declines with age. We evaluated the effects of supplementation with the probiotic Bifidobacterium longum BB536 on immune function and intestinal microbiota in the elderly. MATERIALS AND METHODS In a double-blind study, 45 elderly patients fed by enteral tube feeding (mean [SD] age 81.7 [8.7] years) were given BB536 (n = 23) or a placebo powder (n = 22) for 12 weeks and were observed for an additional 4 weeks posttreatment. At week 4, all patients received an influenza vaccination (A/H1N1, A/H3N2, and B). Clinical data were assessed, including body temperature, bowel movements, fecal microbiota, and immunological biomarkers in blood. RESULTS BB536 intake significantly increased cell numbers of bifidobacteria in fecal microbiota. There was a tendency toward an increase (P = .085 at week 4 and P = .070 at week 16) of serum IgA in the BB536 group compared with the placebo group. BB536 intake did not significantly affect hemagglutination inhibition (HI) titers in response to the influenza vaccine. Natural killer (NK) cell activity decreased significantly in the placebo group during the intervention but not in the BB536 group. Among those subjects with low NK cell activity (<55%, n = 10 for each group), a significant intergroup difference (P < .05) was observed in the changed values from baseline of NK cell activity at weeks 8 and 12. CONCLUSIONS These results shed new light on the potential of long-term ingestion of BB536 in increasing the cell number of bifidobacteria in intestinal microbiota and modulating immune function in the elderly.

Elimination of p19ARF-expressing cells enhances pulmonary function in mice

Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the CDKN2A locus, play critical roles in inducing and maintaining permanent cell cycle arrest during cellular senescence. Although the elimination of p16INK4a-expressing cells extends the life span of the mouse, it is unclear whether tissue function is restored by the elimination of senescent cells in aged animals and whether and how p19ARF contributes to tissue aging. The aging-associated decline in lung function is characterized by an increase in compliance as well as pathogenic susceptibility to pulmonary diseases. We herein demonstrated that pulmonary function in 12-month-old mice was reversibly restored by the elimination of p19ARF-expressing cells. The ablation of p19ARF-expressing cells using a toxin receptor-mediated cell knockout system ameliorated aging-associated lung hypofunction. Furthermore, the aging-associated gene expression profile was reversed after the elimination of p19ARF. Our results indicate that the aging-associated decline in lung function was, at least partly, attributed to p19ARF and was recovered by eliminating p19ARF-expressing cells.

ARF Suppresses Tumor Angiogenesis through Translational Control of VEGFA mRNA

Vascular endothelial growth factor A (VEGFA) is a specific mitogen for vascular endothelial cells that plays a critical role in cancer neoangiogenesis. Here, we report that the nucleolar tumor suppressor p19(ARF) suppresses VEGFA expression, acting at the level of mRNA translation without affecting the transcription of the VEGFA gene. Translational repression of VEGFA mRNA by p19(ARF) does not require p53, a major target of the ARF tumor suppressor pathway, but instead correlates with binding to nucleophosmin/B23. Maintaining VEGFA expression relies on nucleophosmin/B23, and downregulating this protein by RNAi or p19(ARF) leads to translational repression of VEGFA. p19(ARF) inhibits VEGFA-dependent tumor angiogenesis in nude mice. Additionally, p14(ARF) expression and microvessel density are inversely correlated in human colon carcinomas. Taken together, our results define a mechanism by which the ARF tumor suppressor targets the translational repression of specific oncogenes during neoplastic transformation.

Hzf regulates adipogenesis through translational control of C/EBPα

Adipocyte differentiation requires a well‐defined programme of gene expression in which the transcription factor C/EBPα (CCAAT/enhancer‐binding protein) has a central function. Here, we show that Hzf (haematopoietic zinc‐finger), a previously identified p53 transcriptional target, regulates C/EBPα expression. Hzf is induced during differentiation of preadipocyte cell lines, and its suppression by short hairpin RNA disrupts adipogenesis. In Hzfs absence, expression of C/EBPα is severely impaired because of reduced translation of its mRNA. Hzf physically interacts with the 3′ untranslated region of C/EBPα mRNA to enhance its translation. Taken together, these findings underscore a critical role of Hzf in the adipogenesis regulatory cascade.

Cooperative role of the RNA-binding proteins Hzf and HuR in p53 activation

ABSTRACT The RNA-binding protein Hzf (hematopoietic zinc finger) plays important roles in mRNA translation in cerebellar Purkinje cells and adipocytes. We along with others have reported that the expression of the Hzf gene is transcriptionally regulated by the p53 tumor suppressor protein. We show here that Hzf regulates p53 expression in cooperation with HuR. Hzf and HuR independently interact with the 3′ untranslated region (UTR) of p53 mRNA, which facilitates the cytoplasmic localization of p53 mRNA in the presence of the ARF tumor suppressor protein. In the absence of Hzf and HuR, p53 induction by p19ARF is significantly attenuated, and the cells consequently acquire resistance to p19ARF. Thus, these findings demonstrate that in addition to Mdm2 inhibition, p19ARF increases the concentration of p53 through posttranscriptional control of p53 mRNA and suggest critical roles for the RNA-binding proteins Hzf and HuR in p53 induction.

Comparison of the human genomic structure of the Runt domain-encoding PEBP2/CBFα gene family

We cloned the human cDNA corresponding to the cDNA (PEBP2alphaB-451) encoding the mouse polyomavirus enhancer-binding protein 2alphaB-451, representing a major splice variant from acute myeloid leukemia gene 1 (AML1). Genomic DNA clones of AML1 were also isolated and the exon/intron structure was determined. Furthermore, we determined and compared the genomic structures of three mammalian Runt domain-containing genes, PEBP2alphaA,AML/PEBE2alphaB and PEBP2alphaC.