Phorbol ester facilitates apoptosis in murine fibroblasts pretreated by mild ultraviolet radiation.

Although phorbol 12-myristate 13-acetate (PMA) inhibits apoptosis and promotes the growth of some types of cells, it induces apoptosis in other cells. We evaluated the apoptotic effects of PMA on murine fibroblasts (L-929) that had been exposed to ultraviolet-B (UV-B) radiation at 312 nm, which promotes tumor cell growth. Exposure to PMA alone did not induce Fas, Fas-L, or apoptosis. Cells exposed to mild UV-B irradiation (80 J/m(2)) alone exhibited a slight expression of Fas and Fas-L 36 to 48 h after the exposure, and exhibited apoptosis as evidenced by DNA fragmentation 72 h after exposure. The addition of PMA (0.8 x 10(-5) to 3.2 x 10(-5) M) to the medium 24 h after the UV-B exposure markedly and dose-dependently enhanced these cell responses. Confluent untreated cells, cells cocultured with PMA, and cells cocultured with PMA for 24 h after the UV-B exposure consistently expressed mRNAs for wild-type p53, bcl-2, and ICE. Expression of c-myc mRNA was initially observed, but became undetectable in the cells cocultured for 24 h with a high concentration of PMA (3.2 x 10(-5) M) following UV-B exposure. Such cells subsequently exhibited the maximal apoptotic response. We conclude that mild exposure to UV-B altered murine fibroblast cells in such a way as to facilitate their death by apoptosis upon addition of PMA.     

Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

On the mechanism of the peroxidase-catalyzed oxygen-transfer reaction

We reported evidence that horseradish peroxidase (HRP) and chloroperoxidase (CPO) catalyze oxygen transfer from H2O2 to thioanisoles [Kobayashi, S., Nakano, M., Goto, T., Kimura, T., & Schaap, A. P. (1986) Biochem. Biophys. Res. Commun. 135, 166-171]. In the present paper, the reaction mechanism of this oxygen transfer is discussed. The oxidation of para-substituted thioanisoles by HRP compound II showed a large negative rho value of -1.46 vs. the sigma + parameter in a Hammett plot. These results are in accord with the formation of a cation radical intermediate in the rate-determining step. Hammett treatments for HRP- and CPO-dependent S-oxygenations did not provide unequivocal proofs to judge the reaction mechanism, because of the poor correlations for sigma + and sigma p parameters. Different behavior was found in kinetics and stereoselectivity between the two enzymes. Results in the present study and recent studies strongly suggested the formation of a cation radical intermediate. The oxygen atom would transfer by reaction of compound II and the cation radical intermediate. Although involvement of the cation radical was not confirmed in the CPO system, a similar mechanism was proposed for CPO.     

Phenotypic Characterization of Adenovirus Type 12 Temperature-Sensitive Mutants in Productive Infection and Transformation

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxyl-amine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for “initiation” of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutated in H12ts505 is required to maintain at least some aspects of the transformed state.     

Quantitative detection of bisphenol A and bisphenol A diglycidyl ether metabolites in human plasma by liquid chromatography–electrospray mass spectrometry

Due to the ubiquity of epoxy resin compounds and their potential role in increasing the risk for reproductive dysfunction and cancer, the need for an assessment of human exposure is urgent. Therefore, we developed a method for measuring bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) metabolites in human blood samples using high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS). Human blood samples were processed using enzymatic deconjugation of the glucuronides followed by a novel sample preparation procedure using a solid-phase-cartridge column. This selective analytical method permits rapid detection of the metabolites, free BPA and a hydrolysis product of BADGE (BADGE-4OH) with detection limits in the low nanogram per milliliter range (0.1 ng ml⁻¹ of BPA and 0.5 ng ml⁻¹ of BADGE-4OH). The sample extraction was achieved by Oasis HLB column on gradient elution. The recoveries of BPA and BADGE-4OH added to human plasma samples were above 70.0% with a standard deviation of less than 5.0%. This selective, sensitive and accurate method will assist in elucidating potential associations between human exposure to epoxy-based compounds and adverse health effects.     

Demonstration of the presence of G-proteins in hepatic microsomal fraction

The presence of G-proteins in isolated hepatic microsomal vesicles is demonstrated. The G-proteins were identified by their capacity to be ADP-ribosylated by cholera and pertussis toxins. Cholera toxin identified 42 and 45 kDa proteins, corresponding to alpha s-1 and alpha s-2, respectively. Pertussis toxin identified a 40 kDa protein corresponding to alpha i. The microsomal G-proteins are identical to the corresponding G proteins of the plasma membrane, but are present in different proportions; the microsomes have considerably less alpha s proteins than the plasma membrane.     

Influence of rainfall on sleeping site choice by a group of anubis baboons (Papio anubis)

For diurnal nonhuman primates, shifting among different sleeping sites may provide multiple benefits such as better protection from predators, reduced risk of parasitic infection, and closer proximity to spatially and temporally heterogeneous food and water. This last benefit may be particularly important in sleeping site selection by primates living in savanna‐woodlands where rainfall is more limited and more seasonally pronounced than in rainforests. Here, we examined the influence of rainfall, a factor that affects food and water availability, on the use of sleeping sites by anubis baboons (Papio anubis) over two 13‐month study periods that differed in rainfall patterns. We predicted that during wet periods, when food and water availability should be higher, the study group would limit the number of sleeping sites and would stay at each one for more consecutive nights than during dry periods. Conversely, we predicted that during dry periods the group would increase the number of sleeping sites and stay at each one for fewer consecutive nights as they searched more widely for food and water. We also predicted that the group would more often choose sleeping sites closer to the center of the area used during daytime (between 07:00 and 19:00) during wet months than during dry months. Using Global Positioning System data from collared individuals, we found that our first prediction was not supported on either monthly or yearly timescales, although past monthly rainfall predicted the use of the main sleeping site in the second study period. Our second prediction was supported only on a yearly timescale. This study suggests that baboons’ choice of sleeping sites is fluid over time while being sensitive to local environmental conditions, one of which may be rainfall.     

Purification and characterization of glyoxalase I from Pseudomonas putida

Glyoxalase I was purified to apparent homogeneity from Pseudomonas putida. The enzyme was a monomer with a molecular weight of 20,000. The enzyme was most active at pH 8.0. The Km values for methylglyoxal and 4,5-dioxovale-rate are 3.5 mM and 1.2 mM, respectively. Contrary to the case of eukaryotic enzymes, chelating agents showed little inhibitory effects on the enzyme activity. Among the metal ions tested, Zn++ specifically and completely inhibited the activity of the enzyme at a millimolar level. The properties of bacterial glyoxalase I were quite different from mammalian and yeast enzymes.