Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23

The Arf tumor suppressor inhibits cell cycle progression through both p53-dependent and p53-independent mechanisms, including interference with rRNA processing. Using tandem-affinity-tagged p19(Arf), we purified Arf-associated proteins from mouse NIH 3T3 fibroblasts undergoing cell cycle arrest. Tagged p19(Arf) associated with nucleolar and ribosomal proteins, including nucleophosmin/B23 (NPM), a protein thought to foster the maturation of preribosomal particles. NPM is an abundant protein, only a minor fraction of which binds to p19(Arf); however, a significant proportion of p19(Arf) associates with NPM. The interaction between p19(Arf) and NPM requires amino acid sequences at the Arf amino terminus, which are also required for Mdm2 binding, as well as the central acidic domain of NPM and an adjacent segment that regulates NPM oligomerization. The interaction between p19(Arf) and NPM occurs in primary mouse embryonic fibroblasts, including those lacking both Mdm2 and p53. In an NIH 3T3 derivative cell line (MT-Arf) engineered to conditionally express an Arf transgene, induced p19(Arf) associates with NPM and colocalizes with it in high-molecular-weight complexes (2 to 5 MDa). An NPM mutant lacking its carboxyl-terminal nucleic acid-binding domain oligomerizes with endogenous NPM, inhibits p19(Arf) from entering into 2- to 5-MDa particles, and overrides the ability of p19(Arf) to retard rRNA processing.     

Relief effect of vitamin A on the decreased motility of sperm and the increased incidence of malformed sperm in mice exposed neonatally to bisphenol A

Administration of 50 g of bisphenol A (BPA) for the first 5 days after birth resulted in a decrease in the percentage of moving sperm, and an increase in the incidence of malformed sperm, in the epididymides of mice at 10 weeks of age, although no marked changes were found in the testicular histology between BPA-treated and vehicle-treated control mice. The deteriorating effects of 50 g of BPA were ameliorated by the concurrent administration of 100 IU of retinol acetate (RA). Neonatal treatment with 0.5 g of BPA for 5 days resulted in an increase in the incidence of malformed sperm, whereas the BPA effect became more severe in mice nursed by mothers fed a vitamin A-deficient (VAD) diet only a few days before and after parturition. On the other hand, neonatal treatment with 20 g of estrogen for the first 5 days after birth resulted in an increase in the number of estrogen receptor (ER)-positive cells in the epithelium of the vas deferens, whereas only a few epithelial cells showed weak ER-positive signals in the vehicle-treated control mice at 18 days after birth. This increase, however, was suppressed by the concurrent administration of RA. Although five daily treatments with 50 g BPA led to no significant increase in the number of ER-positive cells, it may have been due to the weak estrogenic activity of BPA, as discussed. These findings clearly showed that in mice, neonatal exposure to a relatively large dose of BPA causes damage to the motility and morphology of sperm, but the BPA effect is, to some extent, inhibited by a supplement of VA, and enhanced under a VAD condition.This work was supported by Grants-in-Aid for Encouragement of Young Scientists, Scientific Research (C) and Scientific Research on Priority Areas (A) from the Ministry of Education, Science, Sports, and Culture, Japan     

A novel neurotrophic role of secretory phospholipases A2 for cerebellar granule neurons

Cultured cerebellar granule neurons (CGNs) require membrane depolarization or neurotrophic factors for their survival in vitro and undergo apoptosis when deprived of these survival-promoting stimuli. Here, we show that secretory phospholipases A(2)s (sPLA(2)s) rescue CGNs from apoptosis after potassium deprivation. The neurotrophic effect required the enzymatic activity of sPLA(2)s, since catalytically inactive mutants of sPLA(2)s failed to protect CGNs from apoptosis. Consistently, the ability of sPLA(2)s to protect CGNs from apoptosis correlated with the extent of sPLA(2)-induced arachidonic acid release from live CGNs. The survival-promoting effect of sPLA(2) was inhibited by depletion of extracellular Ca(2+) or by the presence of L-type Ca(2+) channel blocker nicardipine, suggesting that Ca(2+) influx occurs upon sPLA(2) treatment. Among the mammalian sPLA(2)s tested, only group X sPLA(2), but not group IB nor IIA sPLA(2)s, displayed neurotrophic activity. These results suggest a novel, unexpected neurotrophin-like role of sPLA(2) in the nervous system.     

DNA-based biosensor for monitoring pH in vitro and in living cells

DNA is a promising material for the construction of a biosensor or bioindicator because its structure is sensitive to the binding of cofactors. In the current studies, we found that a combination of two DNA oligonucleotides, 5'-TCTTTCTCTTCT-3' and 5'-AGAAAGAGAAGA-3', exhibit a novel structural transition from a Watson-Crick antiparallel duplex to a parallel Hoogsteen duplex as the pH changes from pH 7.0 to 5.0. By labeling this DNA for fluorescence resonance energy transfer, we were able to develop a sensitive pH indicator that can detect changes between pH 7.0 and 5.0. Moreover, using DNA-based hairpin parallel-stranded duplex in conjunction with fluorescence microscopy, we were able to observe the pH changes in living cells during apoptosis as an easily detected change in color. These results indicate that the DNA-based pH indicator should be useful for detecting pH changes between pH 7.0 and 5.0 in living cells.     

The application of phenylmethanethiol and benzenethiol derivatives as odorless organosulfur reagents in the synthesis of thiosugars and thioglycosides

p-octyloxyphenylmethanethiol and p-dodecylbenzenethiol were prepared as new odorless organosulfur reagents. Thiosugars and thioglycosides were synthesized using these reagents without encountering any malodorous procedures.     

Glyceraldehyde-3-phosphate dehydrogenase in the extracellular space inhibits cell spreading

The occurrence and the novel function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the extracellular space were studied. The extracellular GAPDH with the same molecular mass as the intracellular GAPDH was detected in the conditioned medium of mammalian cultured cell lines such as COS-7, HEK293, MCF-7, HepG2, PC-12, and Neuro-2a cells. Western blot analysis represented the occurrence of GAPDH, but not alpha-tubulin (an intracellular marker protein), in the conditioned medium of COS-7 cells. Furthermore, GAPDH was found in rat serum. These results indicate that GAPDH was secreted outside of the cells. Addition of GAPDH to the cultured medium of COS-7, HEK293, and HepG2 cells allowed cells to undergo morphological changes. In COS-7 cells, the extracellular GAPDH inhibited cell spreading without influencing the cell growth. Western blot and immunofluorescent microscopy analyses revealed that the extracellular GAPDH bound to COS-7 cells in time- and dose-dependent manners. However, a mutant substituting Ser for Cys at position 151 of GAPDH resulted in no binding to the cells, no decreased cell-spreading efficiency and no cell morphological changes. These results indicate that the Cys151 was involved in the binding of GAPDH to cells and the GAPDH-inhibited cell spreading.     

Ovotransferrin antimicrobial peptide (OTAP-92) kills bacteria through a membrane damage mechanism

Ovotransferrin antimicrobial peptide (OTAP-92) is a cationic fragment of hen ovotransferrin (OTf). OTAP-92 consists of 92 amino acid residues located within the 109-200 sequence of the N-lobe of OTf. This study was aimed to delineate the antimicrobial mechanism of OTAP-92 and to identify its interaction with bacterial membranes. OTAP-92 caused permeation of Escherichia coli outer membrane (OM) to 1-N-phenylnaphthylamine fluorescent probe in a dose-dependent manner. These results suggested that OTAP-92 crossed the bacterial OM by a self-promoted uptake. Cytoplasmic membrane of E. coli was found to be the target for OTAP-92 bactericidal activity, as assayed by the unmasking of cytoplasmic beta-galactosidase due to membrane permeabilization in a kinetic manner. Pretreatment of bacteria with uncoupler, carbonyl cyanide m-chlorophenylhydrazone, markedly enhanced permeation of cytoplasmic membrane, suggesting that the membrane permeation due to OTAP-92 is independent of the transmembrane potential. In an E. coli phospholipid liposome model, it was demonstrated that OTAP-92 has the ability to dissipate the transmembrane electrochemical potential. Intrinsic fluorescence spectra of the two tryptophan residues in OTAP-92, using liposomal membrane, have identified the lipid-binding region as a helix-sheet motif, and suggested an adjacent Ca(2+)-sensitive site within OTAP-92. These data indicated that OTAP-92 possesses a unique structural motif similar to the insect defensins. Further, this cationic antimicrobial peptide is capable of killing Gram-negative bacteria by crossing the OM by a self-promoted uptake and cause damage to the biological function of cytoplasmic membrane.     

Chediak-Higashi syndrome mutation and genetic testing in Japanese black cattle (Wagyu)

Chediak-Higashi Syndrome (CHS) is an autosomal recessive disorder that affects several species including mice, humans, and cattle. Evidence based on clinical characteristics and somatic cell genetics suggests that mutations in a common gene cause CHS in the three species. The CHS locus on human chromosome 1 and mouse chromosome 13 encodes a lysosomal trafficking regulator formerly known as LYST, now known as CHS1, and is defective in CHS patients and beige mice, respectively. We have mapped the CHS locus to the proximal region of bovine chromosome 28 by linkage analysis using microsatellite markers previously mapped to this chromosome. Furthermore, we have identified a missense A:T-->G:C mutation that results in replacement of a histidine with an arginine residue at codon 2015 of the CHS1 gene. This mutation is the most likely cause of CHS in Wagyu cattle. In addition, we describe quick, inexpensive, PCR based tests that will permit elimination of the CHS mutation from Wagyu breeding herds.