Sequencing of a 23 kb fragment from Saccharomyces cerevisiae chromosome VI

Plasmid clone gapB and lambda phage clone 4682, which contain fragments of Saccharomyces cerevisiae chromosome VI, were analysed. A 23 kb sequence was determined and ten open reading frames (ORFs) were revealed. Among them, five ORFs were identical to five yeast genes (SEC4, MSH4, SPB4, DEG1 and NIC96), two were identical to transposable elements (TYA and TYB), one (gapBorfF003) was highly homologous to a yeast expressed sequence tag, and another (4682orfF002) was predicted to be a nuclear protein. Sequence data have been submitted to DDBJ/EMBL/GenBank data library under Accession Number D44604 (clone gapB) and D44600 (clone 4682), respectively.     

Keratin sponge: immobilization of lysozyme

A porous wool keratin sponge was used for immobilization of lysozyme, a model bioactive substance and was demonstrated to be a unique biomaterial in terms that the activity of lysozyme linked to the sponge through disulfide bond was gradually released, while lysozyme through thioether bond was stably maintained.     

High hydrostatic pressure treatment impairs AcrAB-TolC pump resulting in differential loss of deoxycholate tolerance in Escherichia coli

We reported previously that high hydrostatic pressure-injured stationary phase cells of Escherichia coli K-12 lost their intrinsic deoxycholate tolerance. The AcrAB-TolC multi-drug resistance pump driven by proton motive force has been argued to be responsible for the tolerance to deoxycholate. In this report, we tested the sensitivity of the AcrAB-TolC (three components) pump to high hydrostatic pressure treatment (HPT). E. coli K-12 treated with HPT became sensitive to AcrAB-TolC-specific drugs such as ethidium bromide, but not to tetracycline which is pumped out by a one-component transporter, Tet. Only E. coli K-12 overproducing both AcrAB and TolC exhibited restored tolerance to deoxycholate after HPT but not E. coli overproducing either TolC or AcrAB. These observations strongly suggest that three-component pumps such as AcrAB-TolC are more susceptible to HPT than one-component pumps such as Tet, resulting in the differential loss of deoxycholate tolerance in high hydrostatic pressure-injured E. coli cells.     

Cell trapping, DNA extraction and Molecular Combing in a microfluidic device for high throughput genetic analysis of human DNA

We report on an integrated microfluidic device for cell trapping, DNA extraction and Molecular Combing which can be used for genetic analysis of human DNA. Our results show that it is possible to isolate and linearize the genetic material of a few cells without introducing any manipulation step of DNA molecules, thus increasing dramatically the size of the events that can be studied on the genome. Such extraction strategy is simple and fast, providing a new possibility of high throughput single-cell DNA analysis which should be applicable to cancer research, genome mapping, and genotoxicity studies.     

A sensitive analytical method for six aflatoxins in rainbow trout muscle and liver

A highly sensitive analysis method for six aflatoxins (aflatoxin B?, B?, G?, G?, M? and aflatoxicol) in rainbow trout muscle and liver was developed. Aflatoxins (AFs) were extracted with acetonitrile-water (9 : 1), purified on an immunoaffinity column, and subjected to HPLC with fluorescence detection after post-column photochemical derivatization. The recoveries of AFs at 0.05 μg/kg spiking levels were 71.4-82.4% in muscle and 80.1-93.0% in liver, and the repeatability relative standard deviations (RSDr) were 0.87-4.6% in muscle and 2.0-6.2% in liver. Limits of quantitation (LOQs) and limits of detection(LODs)of AFs were estimated to be 0.004-0.029 μg/kg, and 0.002-0.012 μg/kg, respectively.     

The Effect of Heterozygous Mutation of Adenylate Kinase 2 Gene on Neutrophil Differentiation

Mitochondrial ATP production plays an important role in most cellular activities, including growth and differentiation. Previously we reported that Adenylate kinase 2 (AK2) is the main ADP supplier in the mitochondrial intermembrane space in hematopoietic cells, especially in the bone marrow. AK2 is crucial for the production of neutrophils and T cells, and its deficiency causes reticular dysgenesis. However, the relationship between ADP supply by AK2 and neutrophil differentiation remains unclear. In this study, we used CRISPR/Cas9 technology to establish two heterozygous AK2 knock-out HL-60 clones as models for reticular dysgenesis. Their AK2 activities were about half that in the wild-type (WT). Furthermore, neutrophil differentiation was impaired in one of the clones. In silico analysis predicted that the obtained mutations might cause a structural change in AK2. Time course microarray analysis of the WT and mutants revealed that similar gene clusters responded to all-trans retinoic acid treatment, but their expression was lower in the mutants than in WT. Application of fructose partially restored neutrophil differentiation in the heterozygous knock-out HL-60 clone after all-trans retinoic acid treatment. Collectively, our study suggests that the mutation of N-terminal region in AK2 might play a role in AK2-dependent neutrophil differentiation and fructose could be used to treat AK2 deficiency.     

Influence of extracorporeal shock wave lithotripsy (ESWL) on renal function assessed by 99mTc-DMSA scintigraphy: comparative analysis between ESWL and percutaneous nephroureterolithotripsy (PNL)

99mTc-DMSA renal scintigraphy was utilized to investigate the influence of ESWL on renal function in comparison with that of PNL. In the beginning, the reproducibility of renal uptake rate by the scintigraphy was examined in eleven healthy volunteers under both non-diuretic and diuretic states. The renal uptake rate was shown to be sufficiently reproducible in the same person in the two different trials. However, the differences and the standard deviations were shown to be a few percentages, which were not statistically significant. Changes in the repeated renal uptake rate seem to indicate not only changes of renal function with the treatment but also some technical errors. Herein, to investigate changes in renal function of the therapeutic side, the uptake ratio rate (rate of uptake rate in the therapeutic side/uptake rate in the contral lateral side) was utilized instead of uptake rate. Renal scintigraphy was carried out in 48 patients with unilateral renal stones before and after ESWL or PNL monotherapy or the combined ESWL and PNL therapies. Within one week of treatment, the uptake ratio rate significantly decreased in patients with PNL or the combined ESWL and PNL, although DMSA uptake rate in the therapeutic side did not significantly changes. Neither renal uptake rate nor uptake ratio rate significantly changed after ESWL treatment. There was no significant difference in changes of uptake ratio rate between Siemens Lithostars Plus and the improved Dornier HM-3 lithotriptors. This study indicated that ESWL monotherapy did not affect the uptake ratio rate, although PNL monotherapy and the combined ESWL and PNL therapies may affect the uptake ratio rate to some extent.