Molecular cloning and characterization of plant genes encoding novel peroxisomal molybdoenzymes of the sulphite oxidase family

The Arabidopsis AtMCP and rice OsMCP genes which encode proteins highly homologous to molybdoenzymes of the sulphite oxidase family were isolated and characterized. Both proteins seemed to possess only a molybdenum cofactor as the redox centre, unlike all the other eukaryotic molybdoenzymes. Putative MCP orthologues were identified in 17 plant species, indicating that MCPs are widely distributed over the plant kingdom [corrected]. An analysis using a green fluorescent protein fusion showed that AtMCP possesses a peroxisomal targeting signal at its C-terminus. Putative peroxisomal targeting signals were also found in all plant MCPs, suggesting the existence of a new redox pathway in this organelle.     

Two lignan dimers from bamboo stems (Phyllostachys edulis)

Phyllostadimers A and B, two bis-lignans in which the two lignan units are directly connected by a C-C bond, were isolated from stems of bamboo, Phyllostachys edulis. Their structures were determined on the basis of spectral evidence. In addition, 14 known compounds were also obtained throughout the investigation. Phyllostadimer A significantly inhibited liposomal lipid peroxidation.     

Simple detection of point mutations in DNA oligonucleotides using SYBR Green I

A novel and simple method for detection of mutations in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) is reported. The SYBR Green I is bound specifically with a duplex DNA oligonucleotide (intercalation). This intercalation induces fluorescent emission at 525 nm with excitation at 494 nm. The fluorescence intensity of mismatched oligonucleotides (40-mer) decreases (by more than 13%) in comparison with the perfectly matched oligonucleotides. Moreover, fluorescence measurement of the SYBR Green I can distinguish various types of single-base mismatches, except for the T-G terminal mismatch. The addition of 20% (v/v) formamide, however, to an oligonucleotide solution improved the sensitivity of detection and also enabled the detection of the T-G terminal-mismatch. This detection method requires only a normal fluorescence spectrophotometer, an inexpensive dye and just 50 pmol of sample DNA.     

Establishment of hepatic stem-like cell lines from normal adult porcine liver in a poly-D-lysine-coated dish with nair-1 medium

The existence, origin, and bipotency of the hepatic stem cell (HeSC) have been investigated. However, the isolation and culture of HeSCs from adult liver tissue is not yet well established, and the mechanism by which HeSCs differentiate into mature cells remains unclear. On the other hand, the development of HeSC-isolating and -culturing methods and the in vitro clonal analysis of their mechanism of differentiation are required to enable clinical applications of regenerative medicine in the liver. For the purpose of providing HeSCs for these studies, we attempted to establish an HeSC line from a normal adult porcine liver using a unique culture system, a poly-D-lysine-coated culture dish with NAIR-1 medium (the PDL-NAIR-1 culture system). Moreover, we examined the differentiating capacity of HeSCs in vitro. We demonstrated that it was possible in the culture system that immature epithelial cells capable of proliferating grew selectively into aggregates and that two hepatic stem-like cell lines, PHeSC-A1 and PHeSC-A2, were established. The results from our data suggest that these hepatic stem-like cell lines were capable of self-renewing and differentiating into hepatocytes or biliary epithelial cells and show that the PDL-NAIR-1 culture system offers the immense advantage of isolating and culturing HeSCs from a normal adult liver. Furthermore, because of the ability to use a clonal analysis in vitro, these cell lines are useful for the investigation of various mechanisms in which HeSCs seem to participate and their application in the study of regenerative medicine in the liver.     

Relationship between adrenomedullin and vasopressin-aquaporin system under general anesthesia

AIM: The roles of adrenomedullin (AM) in body fluid balance under general anesthesia were investigated. METHODS: Time course changes in plasma osmolality, AM, arginine vasopressin (AVP), and urinary aquaporin 2 (AQP2) in 17 patients undergoing abdominal surgery under general anesthesia were examined. RESULTS: Increases in plasma AM levels were observed in parallel with increases in the levels of urinary AQP2/creatinine (Cr) before induction and 90 and 180 min after initiation of anesthesia. Significant correlations between plasma AM and urinary AQP2/Cr (r = 0.62, p < 0.0001) as well as urinary AVP/Cr and AQP2/Cr (r = 0.60, p < 0.0001) were uncovered. Multivariate stepwise analysis identified plasma AM as the critical independent factor affecting urinary AQP2/Cr level. CONCLUSION: A novel correlation of AM and AQP2 which overlays an AVP-AQP2 system may play a key role in fluid homeostasis during general anesthesia.     

Investigation on light-addressable potentiometric sensor as a possible cell-semiconductor hybrid

This article reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible biological cell-semiconductor hybrid that will enable us to make an interface between the physical and biological system. To increase the surface potential sensitivity, we used a LAPS structure with single insulator (SiO2) coated with poly-L-ornithine and laminin (PLOL) on Si. Efficient culturing of PC-12 and nerve cells of Lymnaea stagnalis on PLOL-coated Si3N4 and SiO2 was achieved. The thickness of the PLOL layer was found to be about 4 nm by the atomic force microscope (AFM) measurement. Using the advantage of this thin layer of PLOL, we compared the performance of a novel structure to the previously reported "PLOL-coated Si3N4/SiO2/Si" structure. Due to high insulating capacitance, the photocurrent response of the novel LAPS was found to be very steep. As a result, higher sensitivity was achieved. This steepness did not degrade during 10 days when the sensor surface was kept in contact with the cell culture medium and environment. The thickness of PLOL layer, its ability to improve the biological cell adhesion, enhanced sensitivity, and experiment with simulated neural action potential (AP) applied to the novel LAPS show a good promise for LAPS to be a biological cell-semiconductor hybrid.     

A two-hit model for development of multiple endocrine neoplasia type 2B by RET mutations

Multiple endocrine neoplasia (MEN) type 2B mutations have been reported at methionine 918 or alanine 883 in the tyrosine kinase domain of the RET proto-oncogene. Recently, a new combination of two germline missense mutations at valine 804 and tyrosine 806 was identified in a patient with MEN 2B-like clinical phenotypes including medullary thyroid carcinoma, mucosal neuroma, and marfanoid habitus. In this case, valine 804 and tyrosine 806 were replaced with methionine and cysteine, respectively. In the present study, biological activities of RET with these new mutations were compared with those with known MEN 2A or MEN 2B mutations. The transforming activity of RET with the V804M/Y806C mutation was about 8- to 13-fold higher than that of RET with a single V804M or Y806C mutation. Like RET with the M918T or A883F MEN 2B mutation, the transforming activity of RET with the V804M/Y806C mutation was not affected by substitution of phenylalanine for tyrosine 905 that abolished the activity of RET with the MEN 2A mutation. On the other hand, substitution of phenylalanine for tyrosines 864 and 952 drastically diminished the activity of RET with the V804M/Y806C, M918T or A883F mutation, suggesting that these three mutant proteins have similar biological properties.