Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

Effect of active oxygen species on the productivity of torularhodin by Rhodotorula glutinis No. 21

The effect of active oxygen species on the productivity of torularhodin, an effective antioxidant, by soil yeast, Rhodotorula glutinis no. 21, was examined. Methylene blue, methyviologen and AAPH [2,2'-azobis(2-amidinopropane) dihydrochloride] were used as generators of singlet oxygen, superoxide anion radicals and peroxy radicals, respectively. All of them indicated effectiveness at a dose of 1.0 x 10(-10) to 3.0 x 10(-6) M. Addition of these generators to the culture medium had almost no influence on the biosynthesis of beta-carotene, whereas it had marked enhancement on those of torulene and torularhodin. Production of uric acid by xanthineoxidase remained unchanged at a torularhodin concentration of up to 100 microM. This result suggests that torularhodin does not directly affect the productivity of superoxide anions. It has been proved that torularhodin has a more potent effect on the scavenging of peroxyl radicals and inhibits substrate degradation by singlet oxygen more effectively than beta-carotene does. Continuous addition of methylene blue enhanced the torularhodin accumulation more markedly than single addition.     

Numerical prediction of fraction of eutectic phase in Sn−Ag−Cu soldring using the phase-field method

A combination of macroscale solidification simulation and phase-field calculation is employed to predict the volume fraction of the eutectic phase in Sn-4.0 mass% Ag-XCu solder alloys (X=0.5–1.1 mass%). The solidification simulation incorporates the cooling rate in the phase-field simulation. We assume the residual liquid solidifies as eutectic phase when the driving force for the nucleation of Cu₆Sn₅ amounts to a critical value, which is determined based on the experimental data. Though the calculation results depend on the experimental data, the obtained fractions are about 40% for 0.5 mass% Cu and more than 90% for 1.1 mass% Cu alloy, which shows good agreement with the experimental data.     

Selective isopentane formation from CH3OH on a new one-atomic layer ZrO2/ZSM-5 hybrid catalyst

A new one-atomic layer ZrO₂/ZSM-5 hybrid catalyst was prepared by using the repeated reactions between Zr(OC₂H₅)₄ and of the OH groups of the external surfaces of ZSM-5, followed by calcination. The one-atomic layer ZrO₂ attached on the ZSM-5 surface was characterized by means of X-ray diffraction, X-ray fluorescence and EXAFS. The ZrO₂ overlayer is suggested to epitaxially grow on the ZSM-5(001) plane in a [111] direction of tetragonal ZrO₂. The one-atomic layer ZrO₂/ZSM-5 is a unique catalyst which produces selectively isopentane from CH₃OH.     

Inhibition of intimal hyperplasia after vein grafting by in vivo transfer of human senescent cell-derived inhibitor-1 gene

OBJECTIVE: To define the distribution and determinants of cardiovascular disease events among participants undergoing long-term antihypertensive therapy, and to stratify them into risk groups on the basis of pretreatment clinical profiles. DESIGN: A prospective cohort study of participants in a worksite-based antihypertensive treatment program in New York city (1973-1994). PATIENTS: We studied 8690 systematically treated patients who had at least 6 months of follow-up (average of 5.7 years) and, at entry, had had a systolic blood pressure of > or = 160 mmHg or a diastolic blood pressure of > or = 95 mmHg (after 1992 > or = 140/90 mmHg), or had been being administered antihypertensive medication. MAIN OUTCOME MEASURES: Blood pressure and incidence of morbid and mortal cardiovascular events. RESULTS: Blood pressure control (to 140 +/- 3/87 +/- 7 mmHg) was achieved by the first year and maintained through 18 years of therapy. In nearly 50,000 person-years of follow-up, there were 468 cardiovascular disease events [myocardial infarction including revascularization (282), strokes (93), congestive heart failure (30) and other cardiovascular deaths (63)]. Deaths from cardiovascular disease events accounted for 68% of all deaths. Myocardial infarction was most common throughout, but congestive heart failure incidence surpassed stroke incidence after 10 years. A scheme for risk stratification was constructed after analysis of the independent association of baseline factors and incident cardiovascular events. Upon the basis of ease of ascertainment and their demonstrated associations with occurrence of cardiovascular disease during treatment, we selected five pretreatment factors (history of heart attack, stroke, diabetes, age > or = 55 years and pulse pressure > or = 60 mmHg) to stratify patients into four groups. Those with no risk factor had a low risk (n=2999), those with one had a moderate risk (3042), those with two had a high risk (2237), and those with three or more had a very high risk (412). Overall, the unadjusted rates of incidence of cardiovascular disease events per 1000 person-years for patients in very high and low risk groups differed by factors of six and 14 for men and women, respectively. CONCLUSION: These results demonstrate that long-term control of blood pressure can be achieved in a general population. Nevertheless, cardiovascular disease events still accounted for most morbidity and mortality among these 'recovered' hypertensive patients. At entry, on the basis of readily identifiable characteristics, it was possible to stratify patients according to likelihood of subsequent events occurring despite control of blood pressure. This scheme could provide the basis for targeting more aggressive therapy where the potential for further cardioprotection is greatest.     

The characterization of low energy molecular hydrogen ion—induced defects in synthetic diamond by optical absorption

The results of optical absorption analysis of the synthetic diamonds(type Ib) which were implanted with 40 keV molecular hydrogen ions at doses of 10^15-10^17H/cm^2(at 100K),showed that the increase of optical density(OD) of modified layer(-140nm) in UV-VIS region was dependent upon the damage level caused by ion implantation process.The range of relative optical band gap(Er.opt) around 2.0eV suggested that an amorphous carbon network structure like a-C film,which probably contains some localized subtetrabedral-coordinated clusters embedded in the fourflod(sp^3) sites.was tentatively found in this layer,basing on the optical gap of carbon materials.The evolution of Er,opt with ion fluence indicated that no more hydrogenated carbon compositions were produced in as -implanted samples,while the increase of Er,opt with annealing temperature was very similar to that of hydrogen content dependence of Eopt in hydrogenately amorphous carbon(a-C:H):In addition the optical inhomogeneity of type Ib diamond has been revealed by a 2-dimension topograph in transmission mode at λ=430nm。