Mechanism of force generation of a viral DNA packaging motor

A large family of multimeric ATPases are involved in such diverse tasks as cell division, chromosome segregation, DNA recombination, strand separation, conjugation, and viral genome packaging. One such system is the Bacillus subtilis phage phi 29 DNA packaging motor, which generates large forces to compact its genome into a small protein capsid. Here we use optical tweezers to study, at the single-molecule level, the mechanism of force generation in this motor. We determine the kinetic parameters of the packaging motor and their dependence on external load to show that DNA translocation does not occur during ATP binding but is likely triggered by phosphate release. We also show that the motor subunits act in a coordinated, successive fashion with high processivity. Finally, we propose a minimal mechanochemical cycle of this DNA-translocating ATPase that rationalizes all of our findings.     

Cancer risk from chronic exposures to chemicals and radiation: a comparison of the toxicological reference value with the radiation detriment

This article aims at comparing reference methods for the assessment of cancer risk from exposure to genotoxic carcinogen chemical substances and to ionizing radiation. For chemicals, cancer potency is expressed as a toxicological reference value (TRV) based on the most sensitive type of cancer generally observed in animal experiments of oral or inhalation exposure. A dose–response curve is established by modelling experimental data adjusted to apply to human exposure. This leads to a point of departure from which the TRV is derived as the slope of a linear extrapolation to zero dose. Human lifetime cancer risk can then be assessed as the product of dose by TRV and it is generally considered to be tolerable in a 10^–6–10^–4 range for the public in a normal situation. Radiation exposure is assessed as an effective dose corresponding to a weighted average of energy deposition in body organs. Cancer risk models were derived from the epidemiological follow-up of atomic bombing survivors. Considering a linear-no-threshold dose-risk relationship and average baseline risks, lifetime nominal risk coefficients were established for 13 types of cancers. Those are adjusted according to the severity of each cancer type and combined into an overall indicator denominated radiation detriment. Exposure to radiation is subject to dose limits proscribing unacceptable health detriment. The differences between chemical and radiological cancer risk assessments are discussed and concern data sources, extrapolation to low doses, definition of dose, considered health effects and level of conservatism. These differences should not be an insuperable impediment to the comparison of TRVs with radiation risk, thus opportunities exist to bring closer the two types of risk assessment.

     

The respiratory metabolism of a lizard (Lacerta vivipara) in supercooled and frozen states

We investigated the respiratory metabolism of the overwintering lizard Lacerta vivipara while in either supercooled or frozen states. With a variable pressure and volume microrespirometer and a chromatograph, we show that the oxygen consumption of the supercooled animals showed a nonlinear relationship with temperature and an aerobic metabolism demand between 0.5 and -1.5 degrees C. A significant increase in the respiratory quotient (RQ) values indicated an increasing contribution by the anaerobic pathways with decreasing temperature. In the frozen state, two phases are easily detectable and are probably linked to the ice formation within the body. During the first 5-6 h, the animals showed an oxygen consumption of 3.52 +/- 0.28 microl. g(-1). h(-1) and a RQ value of 0.52 +/- 0.09. In contrast, after ice equilibrium, oxygen consumption decreased sharply (0.55 +/- 0.09 microl. g(-1). h(-1)) and the RQ values increased (2.49 +/- 0.65). The present study confirms the fact that supercooled invertebrates and vertebrates respond differently to subzero temperatures, in terms of aerobic metabolism, and it shows that aerobic metabolism persists under freezing conditions.     

Application of isotopic ratio mass spectrometry for the in vitro determination of demethylation activity in human liver microsomes using N-methyl-13C-labeled substrates

The reaction of demethylation mediated by cytochrome P450 (CYP) leads to the equimolar production of demethylated metabolite and formaldehyde. From a 13C-substrate labeled on a carbon of the methyl moiety, [13C]formaldehyde (H13CHO) is liberated. A highly sensitive and specific assay involving the oxidation of H13CHO to 13CO(2) by a double-enzymatic-step reaction is reported. The 13CO(2) was quantified by the method of reverse isotopic dilution based on gas chromatography-isotope ratio mass spectrometry analysis. The method first involves the limiting step of the CYP-dependent reaction, which is stopped with a mixture of zinc sulfate 5 mM and trichloroacetic acid 100 mM. Then, the transformation of H13CHO to 13CO(2) is performed with the formaldehyde (0.2 unit) and the formate (0.2 unit) dehydrogenase NAD-dependent enzymes. The recovery of 13CO(2) from the incubation mixture was equal to 91.4 +/- 3.0%. The accuracy and the precision of the present method were within 12 and 10%, respectively. The limit of quantification was set to 25 pmol. The performance of the assay was validated on human liver microsomes with five probes: [13C]erythromycin, [1-13C]caffeine, [3-13C]caffeine, [7-13C]caffeine, and [13C(2)]aminopyrine. This method is useful for the rapid determination of N-demethylase activity of human liver microsomes from methyl-13C-substrates.     

In vitro tests for haematotoxicity: prediction of drug-induced myelosuppression by the CFU-GM assay

In a prevalidation study, a standard operating procedure (SOP) for human and mouse in vitro tests was developed, for evaluating the potential haematotoxicity of xenobiotics in terms of their direct, adverse effects on the myeloid colony-forming unit (CFU-GM). Based on the adjustment of the mouse-derived maximum tolerated dose (MTD), a prediction model was set up to calculate the human MTD, and an international blind trial was designed to apply this model to the clinical neutropenia of 23 drugs including 17 antineoplastics. The model correctly predicted the human MTD for 20 drugs out of the 23 (87%). This high percentage of predictivity, and the reproducibility of the SOP testing, confirmed the scientific validation of this model, and suggest promising applications for developing and validating other in vitro methods for use in haematotoxicology.     

Validation and use of an enzymic time-temperature integrator to monitor thermal impacts inside a solid/liquid model food

Heat denaturation kinetics of Bacillus licheniformis alpha-amylase, equilibrated at 81% equilibrium relative humidity at 4 degrees C (BLA81), was studied with help of isothermal and nonisothermal conditions by monitoring the decrease in enthalpy associated with the heat denaturation of the enzyme. Due to its low water content, BLA81 denaturation could be studied in the range of 118-124 degrees C. Two batches of BLA81 were successfully validated under nonisothermal conditions allowing the determinations of process values (reference temperature of 121.1 degrees C) in the range of 1-15 min. In a second step, BLA81 was used as a time-temperature integrator (TTI) to investigate potential differences of process values received by freely moving spherical particles as compared to a centrally fixed particle (single-position impact) inside cans containing water as brine. Results showed that the process value received by freely moving particles can be from 5.6% (4 rpm) to 19.7% (8 rpm) smaller than the process value received by the centrally fixed sphere. This means that evaluating the process value by means of a particle fixed at the critical point in a package can lead to potentially overestimations of the actual process value with possible hazardous quality/safety implications. These results highlight the potentials of the TTI technology to monitor the safety of heat-processed agitated solid/liquid foodstuffs.     

Intestinal preconditioning prevents systemic inflammatory response in hemorrhagic shock. Role of HO-1

Intestinal ischemia-reperfusion has been implicated in the systemic inflammatory response and organ injury in hemorrhagic shock, but the exact role of the intestine has never been directly demonstrated. Preconditioning (PC) with brief periods of intermittent ischemia is a known potent anti-ischemic intervention and thus can be used as a tool to assess the role of local intestinal ischemia-reperfusion injury in systemic inflammatory response. Thus rats were first subjected to sham surgery or intestinal preconditioning with four cycles of 1-min ischemia and 10 min of reperfusion 24 h before hemorrhagic shock followed by resuscitation. PC reduced fluid requirements, lung edema, and lactate and tumor necrosis factor-alpha production. These effects were abolished by the heme-oxygenase-1 (HO-1) inhibitor tin protoporphyrin (Sn-PP). PC induced more than fivefold in intestinal HO-1 expression. These results suggest that intestinal ischemia-reperfusion is a major trigger for inflammatory response and organ injury in nonseptic shock. HO-1 appears to play an important role in the protective effect of intestinal preconditioning.