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The use of hair roots as experimental samples has been a research focus for understanding the effects of spaceflight on astronauts, because it has many advantages, one of which is the fact that hair matrix cells actively divide in a hair follicle and sensitively reflect the physical conditions of the human body. In 2009, a research program focusing on the analysis of astronauts’ hairs was initiated to examine the effects of long-term spaceflight on the gene expression and mineral metabolism in the human body. Since the number of samples per astronaut is limited to 5 strands of hairs at each sampling point, due to the ethical viewpoint of astronauts or limited resources in space, it is important to develop an effective method for the molecular analysis of small amounts of hair roots. In this study, mRNA successfully extracted from 1, 5, and 10 hair follicles was amplified and subjected to the DNA microarray analysis to compare the gene expression within subjects. The results indicated that (1) it was possible to perform the genetic analysis on hair samples stored at -80℃, even without a fixation buffer and (2) the newly modified method of mRNA extraction and analysis was effective in detecting differential gene expression in samples containing only 5 hairs. In conclusion, RNA was efficiently extracted from 5 hair roots, which is the same number of hair roots used in the space experiment; therefore, this method can be applied to genetically analyze astronauts’ hair samples.

Accepted: 2016-02-19, 資料番号: SA1160030000

Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and (1)H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris.

An underwater acoustic camera enclosed in a pressure-resistant case was constructed to observe underwater marine animals. This enabled the measurement of the size, shape, and behavior of living marine animals in the detection range up to 240 cm. The transducer array of the acoustic camera was driven by 3.5 MHz ultrasonic signals, and B-mode acoustic images were obtained. Observations were conducted for captive animals in a water tank and for natural animals in a field. The captive animals, including fish, squid and jellyfish, were observed, and a three-dimensional internal structure of animals was reconstructed using multiple acoustical images. The most important contributors of acoustic scattering were the swimbladder and vertebra of bladdered fish, and the liver and reproductive organs of invertebrate animals. In a field experiment, the shape, size, and swimming behavior of wild animals were observed. The possibilities and limitations of the underwater acoustic camera for fishery applications were discussed.

The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPARalpha is mainly expressed in the liver, kidney, heart and muscle. PPARalpha activates fatty acid catabolism, stimulates gluconeogenesis and ketone body synthesis and is involved in the control of lipoprotein assembly. Although PPARalpha is well characterized in the liver, its physiological function is unknown in the kidney. To investigate the intimate function of PPARalpha in the kidney, we analyzed the target gene expression in human metastatic renal cell carcinoma cell line, Caki-1, using small interfering RNA (siRNA) against PPARalpha and real-time RT-PCR methods. We found that some selected genes (long-chain fatty-acid-CoA ligase (FACL1), carnitine palmitoyltransferase 1A (CPT1A), adipose differentiation-related protein (ADRP) and aquaporin 3 (AQP3)) were down-regulated by PPARalpha siRNA. These results suggest that PPARalpha regulates fatty acid metabolism and body water homeostasis in this cell line.

An underwater acoustic camera using 3.5‐MHz ultrasonic waves enclosed in a pressure‐resistant case was constructed to observe underwater marine animals. This enabled the measurement of the size, shape, and behavior of living marine animals including internal organs by B‐mode images in the detection range up to 240 cm. Observations were conducted for captive animals in a water tank and for natural animals in a field. The captive animals, including fish, squid, and jellyfish, were observed, and a three‐dimensional internal structure of animals was reconstructed using multiple acoustical images. The most important contributors of acoustic scattering were the vertebra for nonbladdered fish, and the liver and reproductive organs for invertebrate animals. In a field experiment, the shape, size, and swimming behavior of wild animals were observed. The possibilities and limitations of the underwater acoustic camera for fishery applications were discussed. [Work supported by JSPS Grant‐In‐Aid for Scientific Research.]