Cellular uptake of covalent conjugates of oligonucleotide with membrane-modifying peptide, peptaibol

Using the membrane-modifying peptide, trichorovin-XIIa (TV-XIIa), which is an 11-residual peptaibol isolated from the fungus Trichoderma viride, we synthesized covalent conjugates of 20-mer oligonucleotide with TV-XIIa to examine the potential use of TV-XIIa in cellular delivery. The results indicated that the conjugates were progressively taken up by human lung carcinoma A549 cells. Next, the antisense effects of the conjugates on p53 protein expression were examined. The p53 expression was significantly decreased by ca. 20-50% in the presence of the conjugates upon treatment with the transfection solution at the concentration of 5 μM.     

Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis

The signaling molecule Wnt regulates bone homeostasis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.     

A BAC-based integrated linkage map of the silkworm Bombyx mori

Background

In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps.

Results

We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively.

Conclusion

The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects.     

Crystal structure of the full-length bacterial selenocysteine-specific elongation factor SelB

Selenocysteine (Sec), the 21^st amino acid in translation, uses its specific tRNA (tRNA^Sec) to recognize the UGA codon. The Sec-specific elongation factor SelB brings the selenocysteinyl-tRNA^Sec (Sec-tRNA^Sec) to the ribosome, dependent on both an in-frame UGA and a Sec-insertion sequence (SECIS) in the mRNA. The bacterial SelB binds mRNA through its C-terminal region, for which crystal structures have been reported. In this study, we determined the crystal structure of the full-length SelB from the bacterium Aquifex aeolicus, in complex with a GTP analog, at 3.2-Å resolution. SelB consists of three EF-Tu-like domains (D1–3), followed by four winged-helix domains (WHD1–4). The spacer region, connecting the N- and C-terminal halves, fixes the position of WHD1 relative to D3. The binding site for the Sec moiety of Sec-tRNA^Sec is located on the interface between D1 and D2, where a cysteine molecule from the crystallization solution is coordinated by Arg residues, which may mimic Sec binding. The Sec-binding site is smaller and more exposed than the corresponding site of EF-Tu. Complex models of Sec-tRNA^Sec, SECIS RNA, and the 70S ribosome suggest that the unique secondary structure of tRNA^Sec allows SelB to specifically recognize tRNA^Sec and characteristically place it at the ribosomal A-site.     

Cross-linked protein complex exhibiting asymmetric oxidation activities in the absence of added cofactor

A protein complex (PC) suspension exhibits asymmetric biooxidation activities in the absence of any added cofactor such as NAD(P)⁺ or FAD. It can be extracted from pea protein (PP)‐gel (PP encapsulated with Ca²⁺ alginate gel and aerated in air for several hours) using hot water by rotary shaking and powdered by the following three steps: (1) forming precipitates from the suspension using 30% (w/v) aqueous (NH₄)₂SO₄, (2) crosslinking the precipitates with 0.25% (v/v) GA, and (3) preparing the cross‐linked powder by freeze‐drying. The cross‐linked PC (CLPC) performed asymmetric oxidation of the toward (R)‐isomers of rac‐ 1 and rac ‐2 in 50 mM glycine–NaOH (pH 9.0) buffer/DMSO cosolvent [2.07% (v/v)] with high enantioselectivity; thus, the (S)‐isomers can be obtained in greater than 99% ee from the corresponding rac‐p‐substituted naphthyl methyl carbinol (rac‐ 1 and rac ‐2 ). The CLPC activity was not only competitively inhibited by addition of either 1.0 mM ZnCl₂ or a chelating agent such as 1.0 mM EDTA but also denatured by pretreatments: autoclaving at 121°C (20 min) or using 6.0 M guanidine–HCl containing 50 mM DTT. These results indicated that the PC catalytic process may utilize an electron transfer system incorporating a redox cation (e.g., Fe²⁺ ? Fe³⁺ or Zn). Therefore, the newly introduced CLPC can asymmetrically oxidize the substrates without the addition of any cofactor resulting in a low‐cost organic method. Overall, our results show that the CLPC is an easily prepared, low‐cost reagent that can function under mild conditions and afford stereoselectivity, regioselectivity, and substrate specificity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 953–961, 2012     

Production of Nucleic Acid-Related Substances by Fermentative Processes

Enzymatic studies with Brevibacterium ammoniagenes ATCC 6872 demonstrated that 5-phosphoribose pyrophosphokinase and purinenucleotide pyrophosphorylase were involved in the nucleotide synthesis from purine base by ATCC 6872 and that its actual accumulation from base seemed to take place extracellularly through the action of the salvage enzymes leaked out of cells. Mn²⁺ deficiency and the simultaneous presence of pantothenate and thiamine, essential for efficient nucleotide accumulation, caused the extracellular leakage of the two enzymes with the simultaneous excretion of R5P. In the direct IMP fermentation with the adenine auxotroph, it was verified that hypoxanthine first produced de novo was reconverted into IMP extracellularly by the salvage enzymes as speculated previously.

A guanine-requiring mutant of Brevibacterium ammoniagenes ATCC 6872 accumulated a large amonnt of 5′-xanthosine-monophosphate (abbreviated as XMP).

The quantity of XMP accumulated by the strain was affected significantly by guanine levels in the medium. The suppression of XMP accumulation by an excessive addition of guanine compounds was recovered by the supply of casamino acids in the medium.

An enzyme in the pathway of de novo XMP synthesis, IMP dehydrogenase (IMP: NAD oxidoreductase, EC 1.2.1.14), was repressed and inhibited by guanine compounds.

The facts that an exogenous xanthine was not converted to XMP by the growing cells and that the activity of XMP-pyrophosphorylase was very low or deficient suggest that XMP accumulation by the strain would be probably due to the direct excretion of the nucleotide from the cells.     

Change in Hair Cycle and Hair Length in Nude Mice by Administration of Deuterium Oxide

D₂O increased hair length in Balb/c nu/nu (nude) mice in our previous study although it has an antimitotic effect in cells. To investigate the mechanism of the effect on the hair length, we examined the change by the administration of D₂O in the duration of the hair cycle and the proliferating activity of the hair matrix in relation with hair length in nude mice. The results showed that 20 or 30% D₂O administration did not change the gross structure of the hairs, the proliferative activity and keratinization of the hair matrix cells, but elongated the hair cycle. The duration of the hair cycle increased by the administration of D₂O in a dose-dependent manner over the examined range and these effects were reversible by discontinuation of D₂O. The change in the hair length correlated with the change in the hair-existing phase particularly. We also showed that the mast cell density in the skin, which is related to the hair cycle, increased in the deuterated mice at anagen VI stage which nearly corresponds to the hair-existing phase. The increase in the mast cell density may be related to the increase in the hair cycle duration. These findings indicate that the increase in hair length may be due to the increase in the duration of the hair cycle, in particular, an increase in the hair-existing phase. This study thus suggests that D₂O slows not only short-term cycles such as circadian clock or ultradian clock, but also the hair cycle which is a long-term cycle.     

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the ‘fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.