Signal Sequence and Keyword Trap in silico for Selection of Full-Length Human cDNAs Encoding Secretion or Membrane Proteins from Oligo-Capped cDNA Libraries

We have developed an in silico method of selection of humanfull-length cDNAs encoding secretion or membrane proteins fromoligo-capped cDNA libraries. Fullness rates were increased toabout 80% by combination of the oligo-capping method and ATGpr,software for prediction of translation start point and the codingpotential. Then, using 5'-end single-pass sequences, cDNAs havingthe signal sequence were selected by PSORT (‘signal sequencetrap’). We also applied ‘secretion or membrane protein-relatedkeyword trap’ based on the result of BLAST search againstthe SWISS-PROT database for the cDNAs which could not be selectedby PSORT. Using the above procedures, 789 cDNAs were primarilyselected and subjected to full-length sequencing, and 334 ofthese cDNAs were finally selected as novel. Most of the cDNAs(295 cDNAs: 88.3%) were predicted to encode secretion or membraneproteins. In particular, 165(80.5%) of the 205 cDNAs selectedby PSORT were predicted to have signal sequences, while 70 (54.2%)of the 129 cDNAs selected by ‘keyword trap’ preservedthe secretion or membrane protein-related keywords. Many importantcDNAs were obtained, including transporters, receptors, andligands, involved in significant cellular functions. Thus, anefficient method of selecting secretion or membrane protein-encodingcDNAs was developed by combining the above four procedures.     

Attenuated accumulation of regulatory T cells and reduced production of interleukin 10 lead to the exacerbation of tissue injury in a mouse model of acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a pathological condition that involves diffuse lung injury and severe hypoxemia caused by pulmonary and systemic diseases. We have established a mouse model of severe ARDS, developed by intratracheal injection of α‐galactosylceramide (α‐GalCer), an activator of natural killer T (NKT) cells, followed by LPS. In the present study, we used this model to investigate the regulatory mechanism in the early inflammatory response during acute lung injury. In α‐GalCer/LPS‐treated mice, the number of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and the expression of a Treg cell‐tropic chemokine, secondary lymphoid‐tissue chemokine (SLC), in the lungs was significantly lower than in mice treated with LPS alone. Giving recombinant (r)SLC increased the number of Treg cells in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced the expression of SLC and the accumulation of Treg cells in the lungs of α‐GalCer/LPS‐treated mice, whereas giving recombinant (r)IFN‐γ reduced the number of Treg cells in mice treated with LPS alone. IL‐10 production was significantly lower in α‐GalCer/LPS‐treated mice than in mice treated with LPS alone. Giving rIL‐10 prolonged survival and attenuated lung injury as a result of reduced production of inflammatory cytokines (such as IL‐1β, IL‐6, TNF‐α, and IFN‐γ) and chemokines (including MCP‐1, RANTES, IP‐10, Mig, MIP‐2, and KC) in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced IL‐10 production in α‐GalCer/LPS‐treated mice. These results suggest that the attenuated accumulation of Treg cells may be involved in the development of severe ARDS through a reduction in the synthesis of IL‐10.

     

Binding of the Sialic Acid-binding Lectin,Siglec-9, to the Membrane Mucin,MUC1, Induces Recruitment of β-Catenin and Subsequent Cell Growth

Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, β-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of β-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited β-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway.     

Affinity Maturation of Cry1Aa Toxin to the Bombyx mori Cadherin-Like Receptor by Directed Evolution

Improvement of the activity and insecticidal spectrum of cloned Cry toxins of Bacillus thuringiensis should allow for their wider application as biopesticides and a gene source for gene-modified crops. The insecticidal activity of Cry toxins depends on their binding to the receptor. Therefore, as a model, we aimed to generate improved binding affinity mutant toxins against Bombyx mori cadherin-like receptor (BtR175) using methods of directed evolution with the expectation of insecticidal activity improved mutants. Four serial amino acid residues of ⁴³⁹QAAG⁴⁴² or ⁴⁴³AVYT⁴⁴⁶ of Cry1Aa were replaced with random amino acids and were displayed on the T7 phage for library construction. Through five cycles of panning of the phage libraries using BtR175, 11 mutant phage clones were concentrated, and mutant toxin sequences were confirmed. The binding affinities of the three mutants were 42-, 15-, and 13-fold higher than that of the wild type, indicating that mutants with improved binding affinity to cadherin can be easily selected from randomly replaced loop 3 mutant libraries using directed evolution. We discuss the development of a genetic engineering method based on directed evolution to improve the binding affinity of Cry toxin to receptors.     

Carbon allocation in a Bornean tropical rainforest without dry seasons

To clarify characteristics of carbon (C) allocation in a Bornean tropical rainforest without dry seasons, gross primary production (GPP) and C allocation, i.e., above-ground net primary production (ANPP), aboveground plant respiration (APR), and total below-ground carbon flux (TBCF) for the forest were examined and compared with those from Amazonian tropical rainforests with dry seasons. GPP (30.61 MgC ha^?1 year^?1, eddy covariance measurements; 34.40 MgC ha^?1 year^?1, biometric measurements) was comparable to those for Amazonian rainforests. ANPP (6.76 MgC ha^?1 year^?1) was comparable to, and APR (8.01 MgC ha^?1 year^?1) was slightly lower than, their respective values for Amazonian rainforests, even though aboveground biomass was greater at our site. TBCF (19.63 MgC ha^?1 year^?1) was higher than those for Amazonian forests. The comparable ANPP and higher TBCF were unexpected, since higher water availability would suggest less fine root competition for water, giving higher ANPP and lower TBCF to GPP. Low nutrient availability may explain the comparable ANPP and higher TBCF. These data show that there are variations in C allocation patterns among mature tropical rainforests, and the variations cannot be explained solely by differences in soil water availability.     

Chemical tagging of a drug target using 5-sulfonyl tetrazole

Irreversible modification is one of the most promising strategies to identify cellular receptors of bioactive small molecules. Here we report that receptor proteins can be chemically tagged using a 5-sulfonyl tetrazole probe. 5-Sulfonyl tetrazole easily accepted nucleophilic attack of thiol groups, while 5-sulfinyl tetrazole did not. These functional groups were introduced into probe molecules of a natural product. Cyclosporine A, an immunosuppressant produced by a microbe, was derivatized to possess 5-sulfonyl tetrazole and a tag group, which enabled chemical tagging of cyclophilin A, the cellular receptor of cyclosporine A. Cyclosporine A derivative possessing 5-sulfinyl tetrazole could not tag cyclophilin A. This technique will allow efficient identification of cellular receptors of bioactive small molecules.     

Arabidopsis AtRRP44A Is the Functional Homolog of Rrp44/Dis3, an Exosome Component,Is Essential for Viability and Is Required for RNA Processing and Degradation

The RNA exosome is a multi-subunit complex that is responsible for 3ʹ to 5ʹ degradation and processing of cellular RNA. Rrp44/Dis3 is the catalytic center of the exosome in yeast and humans. However, the role of Rrp44/Dis3 homologs in plants is still unidentified. Here, we show that Arabidopsis AtRRP44A is the functional homolog of Rrp44/Dis3, is essential for plant viability and is required for RNA processing and degradation. We characterized AtRRP44A and AtRRP44B/SOV, two predicted Arabidopsis Rrp44/Dis3 homologs. AtRRP44A could functionally replace S. cerevisiae Rrp44/Dis3, but AtRRP44B/SOV could not. rrp44a knock-down mutants showed typical phenotypes of exosome function deficiency, 5.8S rRNA 3ʹ extension and rRNA maturation by-product over-accumulation, but rrp44b mutants did not. Conversely, AtRRP44B/SOV mutants showed elevated levels of a selected mRNA, on which rrp44a did not have detectable effects. Although T-DNA insertion mutants of AtRRP44B/SOV had no obvious phenotype, those of AtRRP44A showed defects in female gametophyte development and early embryogenesis. These results indicate that AtRRP44A and AtRRP44B/SOV have independent roles for RNA turnover in plants.