Determination of a new podophyllotoxin derivative, TOP-53, and its metabolite in rat plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method was developed for the determination of a new podophyllotoxin derivative, TOP-53 (I), and TOP-53 glucuronide (II) as its major metabolite in rat plasma and urine. For the analysis of I, the sample was chromatographed on a reversed-phase C18 column with electrochemical detection after consecutive two-step liquid-liquid extractions. Compound II was determined as I after enzymatic hydrolysis of II. This method was validated sufficiently with respect to specificity, accuracy, and precision. The limits of quantitation for both I and II were 2 ng/ml in plasma and 10 ng/ml in urine. The method is thus useful for the pharmacokinetic study of I.     

Substitution of an aspartic acid results in constitutive activation of c-kit receptor tyrosine kinase in a rat tumor mast cell line RBL-2H3

The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.     

Genetic analysis of double-strand break repair in Escherichia coli

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.     

Impulse breakdown voltages of triggered multistage vacuum gap

Several sealed-off triggered vacuum gaps are connected in series to improve hold-off voltage. The characteristics of impulse breakdown voltage of these series-connected gaps are investigated experimentally. The sum hold-off voltage of series-connected gaps decreases to a unit hold-off voltage when the maximum value of voltage division ratio across the gaps increases to unity. Self-breakdown probability of the series-connected gaps is always higher than that of a single gap under the same conditions. Hence, stage efficiency of the multistage gap decreases with increasing number of stages. Its value is 90 percent with 2-stage gap and 75 percent with 5-stage gap, respectively, under the same voltage division ratio and the same gap length (2.0 mm) in each stage. Triggered breakdown voltage of 2- or 3- stage gap is several hundred volts when all gaps are triggered simultaneously at the peak of the main impulse wave and a working voltage range is nearly 100 percent in this case. The working voltage range decreases with number of stages. Its value is 45 percent with 3-stage gap and 15 percent with 5-stage gap, respectively, when one triggered gap is fired for switching.     

Chirality-antimicrobial relationship of quaternary ammonium cationics

Optically active and racemic quaternary ammonium cationics containing a chiral alkyl group, such as N,N-dimethyl-N-(1-phenylethyl)alkylammonium bromides were prepared and showed excellent antimicrobial properties against Gram positive and negative bacteria, with some exceptions, and against fungi. The influence of optical isomerism on their antimicrobial properties showed that the antimicrobial properties between racemic and optically active cationics were obscure. The order of activity of substituted alkyl groups was tetradecyl > dodecyl ° hexadecyl.     

An improved cryofixation method: cryoquenching of small tissue blocks during microwave irradiation

The metal contact method of rapid freezing is greatly improved by irradiating the specimen with microwaves at 2.45 GHz for a short period of time (50 ms), while pushing the specimen onto the surface of the copper block cooled by liquid N₂. The microwave irradiation, together with two technical improvements (a light-mass plunger and a recently developed β-gel shock absorber) for preventing bounce, produces a good freezing zone for squid retina, with high reproducibility for each experimental trial, extending from the contact surface to a depth of about 15 μm, which is comparable to the depth obtained by the metal contact method using liquid He in the absence of microwave irradiation. A good freezing zone was also experimentally demonstrated in specimens of rat liver and heart muscle. Microwave irradiation does not have appreciable effects on the ultrastructure of squid retina. The mechanism underlying the improvement in the rapid freezing under the microwave irradiation is discussed.     

Stress intensity factors of microstructurally small crack

The stress intensity factor (SIF) is widely used for evaluating integrity of cracked components. Averaging the anisotropy of each crystal, the macroscopic behavior of polycrystalline materials is isotropic and homogenous in terms of elastic deformation. However, the anisotropic and/or inhomogeneous property influences on the stress field around a crack if the crack size is small in comparison with the grain. Thus, the SIF of the microstructurally small crack may differ from that in the isotropic body. In present study, the effect of anisotropic/inhomogeneous elasticity on the SIF is investigated by using the finite element analysis (FEA). At first, the SIFs of semi-circular crack in a single crystal and a polycrystalline material are calculated. These reveal that the magnitude of SIF is dependent not only on the crystal orientation but also on the deformation constraint by the neighboring crystals. Then, the statistical scatter of SIF due to the random orientation of crystal orientation in a polycrystal is examined by a Monte Carlo simulation.     

Accelerator Analysis of Tributyltin Adsorbed onto the Surface of a Tributyltin Resistant Marine Pseudoalteromonas sp. Cell

Tributyltin (TBT) released into seawater from ship hulls is a stable marine pollutant and obviously remains in marine environments. We isolated a TBT resistant marine Pseudoalteromonas sp. TBT1 from sediment of a ship’s ballast water. The isolate (10^{9.3 ± 0.2} colony-forming units mL⁻¹) adsorbed TBT in proportion to the concentrations of TBTCl externally added up to 3 mM, where the number of TBT adsorbed by a single cell was estimated to be 10^8.2. The value was reduced to about one-fifth when the lysozyme-treated cells were used. The surface of ethanol treated cells became rough, but the capacity of TBT adsorption was the same as that for native cells. These results indicate that the function of the cell surface, rather than that structure, plays an important role to the adsorption of TBT. The adsorption state of TBT seems to be multi-layer when the number of more than 10^6.8 TBT molecules is adsorbed by a single cell.