A single subunit of a heterotrimeric CCAAT-binding complex carries a nuclear localization signal: piggy back transport of the pre-assembled complex to the nucleus

An unresolved question concerns the nuclear localization of the heterotrimeric CCAAT-binding complex, which is evolutionarily conserved in eukaryotic organisms including fungi, plants and mammals. All three subunits are necessary for DNA binding. In the filamentous fungus Aspergillus nidulans the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the HapB, HapC and HapE subunits. Here, by using various green fluorescent protein constructs, a nuclear localization signal sequence (NLS) of the HapB protein was identified, outside of the evolutionarily conserved domain. HapB-EGFP was transported into the nucleus in both DeltahapC and DeltahapE strains, indicating that its NLS interacts with the import machinery independently of the other Hap subunits. In contrast, HapC-EGFP did not enter the nucleus in the absence of HapE or HapB. A similar finding was made for HapE-EGFP, which did not localize to the nucleus in the absence of HapC or HapB. Addition of the HapB-NLS to either HapC or HapE led to nuclear localization of the respective protein fusions, indicating that both HapC and HapE lack a functional NLS. Furthermore, these data strongly suggest that HapC and HapE have first to form a heterodimer and can be transported only as a heterodimer via the HapB protein into the nucleus. Therefore, the HapB subunit is the primary cargo for the import machinery, while HapC and HapE are transported to the nucleus only as a heterodimer and in complex with HapB via a piggy back mechanism. This enables the cell to provide equimolar concentrations of all subunits to the nucleus.     

Identification of the dominant translation start site in the attB1 sequence of the pET-DEST42 Gateway vector

Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the "TTG" in the attB1 site (30 bp upstream of the "ATG" in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes.     

Biosynthesis of Dictyostelium discoideum differentiation-inducing factor by a hybrid type I fatty acid-type III polyketide synthase

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.     

Palindrome-mediated chromosomal translocations in humans

Recently, it has emerged that palindrome-mediated genomic instability contributes to a diverse group of genomic rearrangements including translocations, deletions, and amplifications. One of the best studied examples is the recurrent t(11;22) constitutional translocation in humans that has been well documented to be mediated by palindromic AT-rich repeats (PATRRs) on chromosomes 11q23 and 22q11. De novo examples of the translocation are detected at a high frequency in sperm samples from normal healthy males, but not in lymphoblasts or fibroblasts. Cloned breakpoint sequences preferentially form a cruciform configuration in vitro. Analysis of the junction fragments implicates frequent double-strand-breaks (DSBs) at the center of both palindromic regions, followed by repair through the non-homologous end joining (NHEJ) pathway. We propose that the PATRR adopts a cruciform structure in male meiotic cells, creating genomic instability that leads to the recurrent translocation.     

A novel obligate cultivation mutualism between damselfish and Polysiphonia algae

In cultivation mutualisms, farming animals prepare fields for cultivars, enhance their growth and harvest them. For example, in terrestrial ecosystems, plant-herbivore cultivation mutualisms arose between humans and their crops only relatively recently. We discovered an obligate cultivation mutualism between a damselfish and an alga in a coral reef ecosystem. The damselfish, Stegastes nigricans, manages algal farms through territorial defence against the invading grazers and through weeding of unpalatable algae. As a result, the algal farms of S. nigricans are dominated by one species, Polysiphonia sp. We performed an exhaustive survey of algal assemblages inside and outside the territories of five damselfish species around the Ryukyu Islands, Japan, using molecular and morphological characteristics. Polysiphonia sp. 1 grew exclusively inside the farms of S. nigricans, and never elsewhere. Since only Polysiphonia sp. 1 is harvested and consumed by the damselfish as a staple food, this interdependent relationship is an obligate cultivation mutualism. This is the first record of an obligate plant-herbivore cultivation mutualism in a marine ecosystem. Our data also suggest that three other Polysiphonia species are facultatively mutual with, commensal with, or parasitic on other damselfish species.     

Demecolcine-assisted enucleation for bovine cloning

The present study demonstrated that demecolcine treatment for at least 30 min produces a membrane protrusion in metaphase II-stage bovine oocytes. The maternal chromosome mass is condensed within the protrusion, which makes it easy to remove the maternal chromosomes for nuclear transfer (NT). Maturation promoting factor activity, but not mitogen-activated protein kinase activity, increased up to 30% in oocytes during demecolcine treatment. One normal healthy calf was obtained after transfer of four NT blastocysts produced following demecolcine treatment. Demecolcine treatment did not increase the potential of NT oocytes to develop into blastocysts. The present study demonstrated that chemically-assisted removal of chromosomes is effective for bovine cloning.     

Osteoclast‐specific Dicer gene deficiency suppresses osteoclastic bone resorption

Osteoclasts are unique cells that resorb bone, and are involved in not only bone remodeling but also pathological bone loss such as osteoporosis and rheumatoid arthritis. The regulation of osteoclasts is based on a number of molecules but full details of these molecules have not yet been understood. MicroRNAs are produced by Dicer cleavage an emerging regulatory system for cell and tissue function. Here, we examine the effects of Dicer deficiency in osteoclasts on osteoclastic activity and bone mass in vivo. We specifically knocked out Dicer in osteoclasts by crossing Dicer flox mice with cathepsin K‐Cre knock‐in mice. Dicer deficiency in osteoclasts decreased the number of osteoclasts (N.Oc/BS) and osteoclast surface (Oc.S/BS) in vivo. Intrinsically, Dicer deficiency in osteoclasts suppressed the levels of TRAP positive multinucleated cell development in culture and also reduced NFATc1 and TRAP gene expression. MicroRNA analysis indicated that expression of miR‐155 was suppressed by RANKL treatment in Dicer deficient cells. Dicer deficiency in osteoclasts suppressed osteoblastic activity in vivo including mineral apposition rate (MAR) and bone formation rate (BFR) and also suppressed expression of genes encoding type I collagen, osteocalcin, Runx2, and Efnb2 in vivo. Dicer deficiency in osteoclasts increased the levels of bone mass indicating that the Dicer deficiency‐induced osteoclastic suppression was dominant over Dicer deficiency‐induced osteoblastic suppression. On the other hand, conditional Dicer deletion in osteoblasts by using 2.3 kb type I collagen‐Cre did not affect bone mass. These results indicate that Dicer in osteoclasts controls activity of bone resorption in vivo. J. Cell. Biochem. 109: 866–875, 2010. © 2009 Wiley‐Liss, Inc.     

Novel acyl coenzyme A (CoA): Diacylglycerol acyltransferase-1 inhibitors: Synthesis and biological activities of diacylethylenediamine derivatives

A series of diacylethylenediamine derivatives were synthesized and evaluated for their inhibitory activity against DGAT-1 and pharmacokinetic profile to discover new small molecule DGAT-1 inhibitors. Among the compounds, N-[2-({[1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]carbonyl}amino)ethyl]-6-(2,2,2-trifluoroethoxy)pyridine-3-carboxamide 3x showed potent inhibitory activity and excellent PK profile. Oral administration of 3x to mice with dietary-induced obesity resulted in reduced body weight gain and white adipose tissue weight.