Fluorescent and luminescent fusion proteins for analyses of amyloid beta peptide aggregation.

The amyloid beta (Aß) peptide is regarded as a causative agent of Alzheimer's disease. In this study, fluorescent and luminescent fusion proteins were constructed to analyze Aß aggregation. A system was developed to monitor changes in luminescence that provides information about Aß aggregation. In the presence of monomeric Aß, the fusion protein exhibits higher luminescence intensity, and the luminescence intensity is diminished after aggregation of the fusion protein and Aß. In contrast, the fluorescence is sustained in the presence of Aß. In the absence of Aß, the fusion protein self-aggregates, and its luminescence and fluorescence are quenched, thus decreasing the background fluorescence and enhancing the detection of Aß inside and outside the cells. The ratio of the luminescence intensity to the fluorescence intensity would allow the aggregation degrees of Aß to be distinguished. This study would be a promising method for analyzing the aggregation state of a particular amyloid protein/peptide (monomer, oligomer, or fibril), as well as the distribution of the amyloid protein/peptide within and at the cell surface, by using a single fusion protein. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Targeting of EGF-displayed protein nanoparticles with anticancer drugs.

The development of protein-based carriers for drug delivery has been well studied. We previously constructed a protein-based nanoparticle consisting of genetically engineered elastin-like polypeptides (ELPs) with a fused poly-aspartic acid tail (ELPD ). The size of the self-assembled ELPD nanoparticles was regulated by charged repulsion of the poly-aspartic acid chains. In the present study, epidermal growth factor (EGF) was genetically fused to the C-terminus of ELPD to impart an active targeting ability to the ELPD nanoparticles. We examined the nanoparticle formation with EGF as well as its targeting ability. ELPD with fused EGF was found to form nanoparticles that displayed multivalent EGFs on their surface. EGF-displayed nanoparticles loaded with the anti-cancer drug paclitaxel were internalized into cells overexpressing the EGF receptor, and induced cell death.

Genetic PEGylation.

Polyethylene glycol (PEG) was genetically incorporated into a polypeptide. Stop-anticodon-containing tRNAs were acylated with PEG-containing amino acids and were then translated into polypeptides corresponding to DNA sequences containing the stop codons. The molecular weights of the PEG used were 170, 500, 700, 1000, and 2000 Da, and the translation was confirmed by mass spectrometry. The PEG incorporation ratio decreased as the molecular weight of PEG increased, and PEG with a molecular weight of 1000 Da was only slightly incorporated. Although improvement is required to increase the efficiency of the process, this study demonstrates the possibility of genetic PEGylation.

[Sequential changes in inflammatory and stress responses during 24-hour running].

Running for an extended period of time can cause severe stress on the body, subsequently damaging skeletal muscle and resulting in changes in blood components. However, few reports have examined vital responses during and after running. This study analyzed inflammatory responses during and after running and changes in stress responses as determined by serial changes in blood components. Venous blood was obtained before starting, 6 h after starting, 12 h after starting, and immediately after finishing 24 h of continuous running. Samples were analyzed for high-sensitivity C-reactive protein (hsCRP), pentraxin 3 (ptx3), white blood cells (WBC), myoglobin, creatine kinase (CK), and hormones. Diet and physical activity were standardized 24 h before and after running. Subjects comprised 16 men who agreed to participate in experimental running on November 8 and 9, 2008, at Tokyo Gakugei University. Mean running distance was 151.32 +/- 32.1 km (range, 83.6-210.0 km) in 24 h. A significant increase in hsCRP was seen from 12 h after starting to completion. Compared to hsCRP, ptx3 gradually increased from before starting to after completion, showing a significant difference between pre and post-run ptx3 levels. WBC count increased significantly until 6 h after starting. Neutrophils in leukocytosis increased significantly during the first 6 h. Eosinophils decreased significantly over the course of the 24 h. Cortisol increased, and testosterone decreased significantly from 6 h after starting. Dehydroepiandrosterone sulfate (DHEA-S), myoglobin, and CK increased over the course of the 24 h. Reactive oxygen metabolites (d-ROMs) changed within the normal range though there was a significant decrease, and biological anti-oxidant potential (BAP) stabilized. Active natural killer cells decreased significantly after 24 h running. Biopyrrin (BPn) increased significantly. Changes in stress oxide were small both during and after running, and adaptation for antioxidation was good. DHEAS, a biomarker of aging, was found to increase over the course of the 24 h, suggesting that controlling decreases in DHEA-S may be possible using exercise, particularly in males. The key finding was that DHEA S levels tended to increase with continuous aerobic exercise.

Direct detection of RNAs in living cells using peptide-inserted Renilla luciferase.

In this study, non-engineered RNAs were detected in living cells using bioluminescence. Two types of probe were utilized: a peptide inserted RLuc (PI-RLuc) probe and a split-RNA probe. Incorporation of the PI-RLuc and split-RNA probes enabled the direct detection of RNA introduced into living cells.

RNA detection using peptide-inserted Renilla luciferase.

A novel complementation system with short peptide-inserted-Renilla luciferase (PI-Rluc) and split-RNA probes was constructed for noninvasive RNA detection. The RNA binding peptides HIV-1 Rev and BIV Tat were used as inserted peptides. They display induced fit conformational changes upon binding to specific RNAs and trigger complementation or discomplementation of Rluc. Split-RNA probes were designed to reform the peptide binding site upon hybridization with arbitrarily selected target RNA. This set of recombinant protein and split-RNA probes enabled a high degree of sensitivity in RNA detection. In this study, we show that the Rluc system is comparable to Fluc, but that its detection limit for arbitrarily selected RNA (at least 100 pM) exceeds that of Fluc by approximately two orders of magnitude.

[Clinical biochemical evaluation of central fatigue with 24-hour continuous exercise].

OBJECTIVE: The present study was conducted to clarify the effects of ultra-marathon (ultra long-term aerobic exercise in which people run long distances) on the brain; examine the issue of central fatigue; verify the serotonin hypothesis of exercise-induced brain fatigue, and ascertain relationships between central fatigue and oxidative stress. METHODS: Subjects consisted of 15 individuals (12 men, 3 women) who ran continuously for 24 h. Mean age was 44 +/- 9 years (range, 31 approximately 64 years). Blood tests were conducted: (1) before starting to run (around 09:00); (2) 16h after starting (02:00 the next day); and (3) just after the finish (around 10:00 the next day) to measure the serum levels of serotonin, melatonin, free tryptophan (f-Tp) and free fatty acid. At the same time, urine samples were collected to measure levels of urinary biopyrrins (BPn). Subjective symptoms were investigated using the Japanese version of the Profile of Mood States (POMS) instrument. RESULTS: (1) Participants ran a mean (+/- SD) distance of 162.6 +/- 18.3 km. (2) There were not marked changes in serum serotonin levels. Serum melatonin levels at 3 time points were 3.4 +/- 0.6 pg/ml, 57.2 +/- 15.2pg/ml and 7.8 +/- 8.9pg/ml, respectively(p < 0.01 before start vs. 16h after start). Serum f-Trp levels at the 3 time points were 5.4 +/- 0.9 nmol/ml, 9.7 +/- 2.1 nmol/ml and 11.5 +/- 4.9 nmol/ml, respectively (p< 0.05 before start vs. just before finish). Free fatty acid levels were 0.42 +/- 0.10 nmol/ml, 1.26 +/- 0.11 nmol/ml and 1.39 +/- 0.23 nmol/ml, respectively (p < 0.01 before start vs. 16 hours after start) (p < 0.05 before start vs. just after finish). (3) Urinary BPn levels increased with time, from 1.2 +/- 0.7 nmol/ml to 2.6 +/- 1.0 nmol/ml to 4.0 +/- 1.5 nmol/ml, respectively (p < 0.01 before the start vs. 16 hours after the start). (4) In terms of POMS scores, fatigue score (Factor F) increased, but vitality score (Factor V) was high at all time points and did not demonstrate any marked changes. Scores for anger and hostility were low (Iceberg profile-type: convex type). Urinary BPn levels were correlated significantly with both serum f-Trp level and Factor F:(y = 8.41x + 2.5, r = 0.708, n = 42) and (y = 2.82x + 5.9, r = 0.568, n = 42), respectively. Urinary BPn thus reflected the degree of subjective fatigue with a high level of sensitivity. CONCLUSIONS: The present results suggest that running continuously for 24h induces brain fatigue and that oxidative stress may be involved.

Emergency image transfer system through a mobile telephone in Japan: technical note.

OBJECTIVE: We report the first application and usefulness of image transfer through a mobile telephone, which can be used between the hospital and another location. METHODS: With this system, brain computed tomographic scans, magnetic resonance images, and angiograms obtained from more than 100 patients in the emergency department were transferred and diagnostically analyzed via a mobile phone. The mobile phone had a 110,000-pixel digital camera and a built-in thin-film transistor liquid crystal display. We reviewed the distribution of pathological characteristics on the transferred images and compared them with the diagnoses made using full-scale original film. RESULTS: This system of transferring images was useful in all cases for correct early diagnosis and early treatment. The quality of magnetic resonance images received was better than that of computed tomographic scans. Hemorrhage provided better contrast than infarction for allowing easy identification. Cerebral angiography revealed small aneurysms if the target area was focused properly. Image quality was sufficient for interpretation despite the small dimensions of the monitor. Ease of operation and portability were both satisfactory. The mean time from commencement of image uptake to complete reception was 2 to 3 minutes. CONCLUSION: The mobile phone system is adequately useful for early diagnosis and initiation of treatment in emergent cases. This is attributable to its low cost and ease of handling for sending images to remote areas and between hospitals, despite the small dimensions of the monitor. Better-quality image transfer will be realized through an advanced mobile phone system based on the new International Mobile Telecommunication 2000 standards, which also will be useful in the development of telemedicine and telecare.

[Absorbed dose in CT: comparison by CT dose index].

Few reports have discussed the absorbed dose on CT units with increased scanning capacity even with the current widespread adoption of multi-slice CT units. To compare and investigate the dose indexes among CT units, we measured the absorbed dose on CT units operating in Nagano Prefecture Japan. The measurements showed proportionality between phantom absorbed dose and the exposured mAs values in conventional scanning operation. Further, the measurements showed that the absorbed dose in the center of the phantom differed by about 2.1-fold between the highest and lowest levels on individual CT units. Within a single company, multi-slice CT units of the same company gave absorbed doses of about 1.3 to 1.5 times those of conventional single-slice CT units under the same exposured conditions of conventional scanning. When the scanning pitch was reduced in helical scanning, the absorbed dose at the center of the phantom increased.