Industrially produced trans-fatty acids are potent promoters of DNA damage-induced apoptosis.

trans-Fatty acids (TFAs) are unsaturated fatty acids harboring at least one carbon-carbon double bond in trans configuration, which are categorized into two groups according to their origin: industrial and ruminant TFAs, hereafter called iTFAs and rTFAs, respectively. Numerous epidemiological studies have shown a specific link of iTFAs to various diseases, such as cardiovascular and neurodegenerative diseases. However, there is little evidence for underlying mechanisms that can explain the specific toxicity of iTFAs, and how to mitigate their toxicity. Herein, we show that iTFAs, including elaidic acid (EA) and linoelaidic acid, but not rTFAs, facilitate apoptosis induced by doxorubicin (Dox), triggering DNA double-strand breaks. We previously established that EA promotes Dox-induced apoptosis by accelerating c-Jun N-terminal kinase (JNK) activation through mitochondrial reactive oxygen species (ROS) overproduction. Consistently, iTFAs specifically enhanced Dox-induced JNK activation. Furthermore, Dox-induced pro-apoptotic signaling by iTFAs was blocked in the presence of oleic acid (OA), the geometrical cis isomer of EA. These results demonstrate that iTFAs specifically exert their toxicity during DNA damage-induced apoptosis, which could be effectively suppressed by OA. Our study provides evidence for understanding the difference in toxic actions between TFA species, and for new strategies to prevent and combat TFA-related diseases.

Anti-survivin single-domain antibodies derived from an artificial library including three synthetic random regions by in vitro selection using cDNA display.

Single-domain antibodies (variable domain of the heavy chain of a heavy chain antibody; VHH) are promising reagents for therapeutics and diagnostics because of their stability, cost-effective production and material workability as a small antibody. Currently, general acquisition of a VHH using immunization of camelids is inconvenient from the standpoint of animal protection, cost and the process is time-consuming. Thus, a straightforward and efficient method for screening VHHs against a target molecule is required. In this study, we examined whether in vitro selection of a VHH against a target protein could be performed by a cDNA display method with an artificial VHH library that had the three complementarity-determining regions (CDRs) randomized by chemical synthesis. The results revealed that a particular VHH against survivin, which is a member of the inhibitor of apoptosis family, was selected with affinity in the range of 10-7 to 10-8 M. The in vitro selection of a VHH using cDNA display with an artificial synthesized library without animal immunization was shown to be effective for rapid and inexpensive screening of VHHs against a target protein.

CD27(-)CD45(+) γδ T cells can be divided into two populations, CD27(-)CD45(int) and CD27(-)CD45(hi) with little proliferation potential.

In addition to the majority of T cells which carry the αß T cell receptor (TCR) for antigen, a distinct subset of about 1-5% of human peripheral blood T cells expressing the γδ TCR contributes to immune responses to infection, tissue damage and cancer. T cells with the Vδ2(+) TCR, usually paired with Vγ9, constitute the majority of these γδ T cells. Analogous to αß T cells, they can be sorted into naive (CD27(+)CD45RA(+)), central memory (CD27(+)CD45RA(-)), effector memory (CD27(-)CD45RA(-)), and terminally-differentiated effector memory (CD27(-)CD45RA(+)) phenotypes. Here, we found that CD27(-)CD45RA(+) γδ T cells can be further divided into two populations based on the level of expression of CD45RA: CD27(-)CD45RA(int) and CD27(-)CD45RA(hi). Those with the CD27(-)CD45RA(hi) phenotype lack extensive proliferative capacity, while those with the CD27(-)CD45RA(int) phenotype can be easily expanded by culture with zoledronate and IL-2. These CD27(-)CD45RA(hi) potentially exhausted γδ T cells were found predominantly in cancer patients but also in healthy subjects. We conclude that γδ T cells can be divided into at least 5 subsets enabling discrimination of γδ T cells with poor proliferative capacity. It was one of our goals to predict the feasibility of γδ T cell expansion to sufficient amounts for adoptive immunotherapy without the necessity for conducting small-scale culture tests. Fulfilling the ≥1.5% criterion for γδ T cells with phenotypes other than CD27(-)CD45RA(hi), may help avoid small-scale culture testing and shorten the preparation period for adoptive γδ T cells by 10 days, which may be beneficial for patients with advanced cancer.

Role of messenger RNA-ribosome complex in complementary DNA display.

In vitro display technologies such as ribosome display and messenger RNA (mRNA)/complementary DNA (cDNA) display are powerful methods that can generate library diversities on the order of 10(10-14). However, in mRNA and cDNA display methods, the end use diversity is two orders of magnitude lower than initial diversity and is dependent on the downstream processes that act as limiting factors. We found that in our previous cDNA display protocol, the purification of protein fusions by the use of streptavidin matrices from cell-free translation mixtures had poor efficiency (∼10-15%) that seriously affected the diversity of the purified library. Here, we have investigated and optimized the protocols that provided remarkable purification efficiencies. The stalled ribosome in the mRNA-ribosome complex was found to impede this purification efficiency. Among the various conditions tested, destabilization of ribosomes by appropriate concentration of metal chelating agents in combination with an optimal temperature of 30°C were found to be crucial and effective for nearly complete isolation of protein fusions from the cell-free translation system. Thus, this protocol provided 8- to 10-fold increased efficiency of purification over the previous method and results in retaining the diversity of the library by approximately an order of magnitude-important for directed evolution. We also discuss the possible effects in the fabrication of protein chips.

Improvement of a puromycin-linker to extend the selection target varieties in cDNA display method.

cDNA display using a puromycin-linker to covalently bridge a protein and its coding cDNA is a stable and efficient in vitro protein selection method. The optimal design of the often-used puromycin-linker is vital for effective selection. In this report, an improved puromycin-linker containing deoxyinosine bases as cleavage sites, which are recognized by endonuclease V, was introduced to extend the variety of the selection targets to molecules such as RNA. The introduction of this linker enables efficient in vitro protein selection without contamination from RNase T1, which is used for the conventional linker containing ribonucleotide G bases. In addition, mRNA-protein fusion efficiency was found to not depend on the length of the flexible poly (ethylene glycol) (PEG) region of the linker. These findings will allow practical and easy-to-use in vitro protein selection by cDNA display.

[Breast deformities caused by the operation for congenital heart disease].

Breast deformities are seen as one of the late postoperative complications in thoracotomy, but there are very few reports in the literature. We investigated causes and treatments in 5 patients who have consulted to our department after operations for congenital heart disease between April 1989 and March 2011. The injured breast bud in the cardiac operation resulted in hypoplastic deformities, and deformities became apparent during puberty with breast growth. These patients required release of the scar contracture to lead to normal breast development, and also have to been treated chest deformity. After stopping at breast growth in adolescence, some augmentations were necessary if bilateral asymmetry had been remained. Breast deformities are very important issue in the viewpoints of patients' quality of life (QOL), though not relating directly to vital prognosis. We'd like to introduce these complications and some choices about breast reconstruction to thoracic surgeons.

How to reconstruct a natural and deep umbilicus: three methods of umbilicoplasty for five types of umbilical deformities.

UNLABELLED: Conventional methods of umbilicoplasty using V-Y advancement flap often result in unnatural wide or shallow umbilical depressions facing upward or downward. Moreover, although the umbilical deformities have many variations, no report has described the selection of an umbilicoplasty method for types of umbilical deformity. To resolve these problems, we devised 3 methods of umbilicoplasty. In this report, we classified all kinds of umbilical deformities into 5 types, and studied the most suitable method for each type of umbilical deformity. METHOD: The umbilical deformities are classified into Type 0: the defect of umbilicus; Type I, the low-grade protrusion; Type II, the high-grade protrusion with wide base; Type III, the high-grade protrusion with narrow base; and Type IV, the protrusion in depression. The most suitable method among our 3 methods was adapted to each type. Method 1 with a S-shaped skin incision was adapted to Type 0 and I, Method 2 with fan-style flaps was adapted to Type II, and Method 3 with dividing the umbilical protrusion was adapted to Type III and IV. RESULTS: Sixty-three patients (10 cases of Type 0, 31 cases of Type I, 10 cases of Type II, 5 cases of Type III, and 7 cases of Type IV) underwent umbilicoplasty using the suitable method, and all were well corrected. CONCLUSIONS: Using the best choice among our 3 methods, it is easy to create a natural, vertically long and deep umbilical depression without conspicuous scars in all types of umbilical deformities.