Analysis of Cre-loxP interaction by surface plasmon resonance: influence of spermidine on cooperativity

To study target site selectivity of one important class of DNA-binding proteins, site-specific DNA recombinases, we developed an automated real-time kinetic assay based on surface plasmon resonance (BIACORE) and formulated a curve-fitting model that takes into account cooperative interactions. Monitoring the interaction between the Cre DNA recombinase and its specific target site loxP by BIACORE, we found that Cre associates with loxP tightly and highly cooperatively. We observed that the cooperative moment of the Cre-loxP interaction is strongly dependent on the concentration of spermidine, a small polyamine influencing DNA conformation. Thus, DNA conformation can have a profound impact on substrate recognition and subsequent recombination.     

Biosensor detection of triplex formation by modified oligonucleotides

Due to the instability of DNA oligonucleotides in biological solutions, antisense or antigene therapies aimed at modulation of specific gene expression will most likely require the use of oligonucleotides with modified backbones. Here, we examine the use of a surface plasmon resonance biosensor (BIAcore) to compare triplex-directed binding of modified oligonucleotides targeted to a region of the murine c-myc promoter. We describe optimization of experimental conditions to minimize nonspecific interactions between the oligonucleotides and the sensor chip surface, and the limitations imposed by certain backbones and sequence types. The abilities of pyrimidine oligonucleotides with various modified backbones to form specific triple helices with an immobilized hairpin duplex were readily determined using the biosensor. Modification of the third-strand oligonucleotide with RNA or 2(')-O-methyl RNA was found to enhance triplex formation, whereas phosphorothioate or phosphotriester substitutions abrogated it. A comparison of these results to DNase I footprinting experiments using the same oligonucleotides showed complete agreement between the two sets of data.     

Activation of microsomal epoxide hydrolase by interaction with cytochromes P450: kinetic analysis of the association and substrate-specific activation of epoxide hydrolase function

The kinetics of the association between cytochrome P450 (P450) and microsomal epoxide hydrolase (mEH) was studied by means of resonant mirror based on the principle of surface plasmon resonance. The dissociation equilibrium constants (K(D)) for the affinity of P450 enzymes for mEH were estimated by resonant mirror using an optical biosensor cell covalently bound to rat mEH. Comparable K(D) values were obtained for CYP1A1 and 2B1, and these were greater by one order of magnitude than that for the CYP2C11. To clarify the influences of P450 enzymes on the catalytic activity of mEH, the hydrolyzing activity for styrene oxide and benzo(a)pyrene-7,8-oxide [B(a)P-oxide] was analyzed in the presence or absence of P450s. Styrene oxide hydrolysis was activated by all P450s including the CYP1A, 2B, 2C, and 3A subfamilies. In agreement with the association affinity determined by resonant mirror, CYP2C11 tends to have enhanced activity for styrene oxide hydrolysis. On the other hand, B(a)P-oxide hydrolysis was enhanced by only CYP2C11 while CYP1A1 and CYP2B1 had no effect. These results suggest that (1) many P450 enzymes associate nonspecifically with mEH, (2) the CYP2C11 plays a greater role in the association/activation of mEH and (3) the P450-mediated activation of mEH depends upon the substrate of mEH.     

Probing functionalized gold colloids for single particle tracking experiments

Functionalized submicroscopic particles are currently used to label proteins or lipids at the surface of living cells for single particle tracking experiments. In many cases, it can be of crucial importance for the particle to be anchored to a single molecule. We have addressed this question for the labeling at the plasma membrane of NRK cells of the mu-opioid receptor bearing a T7 epitope at the N-terminus. Using biophysical methods we were able to prepare quasi-monovalent anti-T7 antibody conjugated gold colloids (40 nm diameter) leading to stable and specific binding to the receptor. The rational method, we report here, can be extended to design customized probes for the labeling of various tagged molecules.     

The effects of cleaning and disinfection in reducing Salmonella contamination in a laboratory model kitchen

AIMS: To establish a laboratory model to compare the effectiveness of detergent-based disinfection procedures for reducing cross-contamination risks during handling of contaminated chicken. METHODS AND RESULTS: During handling of chickens, artificially contaminated with Salmonella enteritidis PT4, the organism was widely spread to hands, cloths, and hand- and food-contact surfaces. Hygiene procedures were assessed on the basis of their ability to reduce the number of recoverable salmonellas to <1 CFU. Although detergent-based cleaning using a typical bowl-wash routine without rinsing produced some risk reduction (from 100 to 61.4% of contaminated surfaces), it was insufficient to consistently restore surfaces to a hygienic state. By combining detergent-based cleaning with a rinsing step or with hypochlorite at 500 ppm (of available chlorine) some further reduction in microbial risk was achieved, but was not considered satisfactory for food hygiene purposes. By contrast the risk reduction produced by hypochlorite at 5000 ppm was highly significant and was sufficient to reduce the number of contaminated surfaces to 2.9%. CONCLUSIONS: A key step in achieving a hygienic state through detergent-based cleaning is rinsing but even this will not produce a 'hygienic' result for difficult surfaces such as the chopping board or the dishcloth. Disinfectant compounds should be considered in order to reduce the potential for foodborne cross infection within the home environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Although tests are available to determine the performance of disinfectants, there are no quantitative procedures available to compare the risk reduction achieved by disinfection with that produced by detergent-based procedures. This study describes a reproducible laboratory method which can be used to differentiate the effectiveness of different hygiene procedures for reducing cross-contamination risks during food handling.     

Sources of dissolved methane in Lake Biwa

The spatial distribution and seasonal variation in the concentration and carbon isotopic composition of dissolved methane in a river–lake ecosystem were studied in Lake Biwa, Japan, and its tributary rivers. Methane concentrations in all subsystems examined were supersaturated with respect to the atmosphere. The epilimnion showed higher concentrations of dissolved methane than the hypolimnion in the pelagic zone. Peak methane concentrations were observed at the thermocline. The largest amount of methane in the pelagic water column was recorded at the end of a stagnant period, at which the bottom water of the sublittoral zone (30m in depth) exhibited increased methane concentration. Transect observation of dissolved methane revealed three methane peaks at different water depths in the lake, and river water and the sediments in littoral and sublittoral zones were suggested to be the corresponding sources. Water at the river mouth was replete with dissolved oxygen but also contained a high concentration of methane. The present results suggest that river water and littoral sediment are potential sources of dissolved methane in Lake Biwa, and other sources, such as internal waves, are responsible for increased methane in the pelagic zone at the end of stagnant periods. Carbon stable isotope analysis indicated that there were different sources of dissolved methane, although it was difficult to identify the origins due to high variation of the isotopic composition of methane from different sources.     

Variations in lipophilic and vacuolar flavonoids among European Pulicaria species

Four European Pulicaria species, P. odora, P. paludosa, P. sicula and P. vulgare, were analysed for their surface and vacuolar constituents for comparison with previous data obtained for P. dysenterica. Each species had a distinct flavonoid pattern with notable differences between leaf and inflorescence. 6-Hydroxyflavonols were the major lipophilic components in all of the species and tissues except in the leaves of P. paludosa and P. vulgare, where scutellarein 6-methyl ether was the main constituent. In the leaves of P. sicula a more unusual flavone, 6-hydroxyluteolin 5,6,7,3',4'-pentamethyl ether, was a major component. Pulicaria odora was distinguished by the presence of a series of methylated 6-hydroxykaempferol derivatives including a 3,5,6,7,4'-pentamethyl ether. Quercetagetin hexamethyl ether occurred in both tissues of P. sicula together with the 3,7,3,4'-tetra methyl ether and other quercetagetin derivatives, which were 5-methylated. Quercetagetin 3,7,3'-methyl ether was present in all species except P. odora. Flavonol glucuronides were characteristic vacuolar constituents of all the taxa studied. Two rare glycosides, patuletin and 6-hydroxykaempferol 6-methyl ether 7-glucuronides were identified in the inflorescence of P. odora. Pulicaria vulgaris, a rare plant of southern England, had the vacuolar flavonoid profile most similar to the other more abundant British plant, P. dysenterica.     

Lymphocytes apoptosis: young versus aged and humans versus rats

This paper deals with a comparative study of lymphocyte apoptosis in young versus aged and humans versus rats. Apoptotic rate achieved by the use of different apoptogenic inducers, acting at different cellular levels, and cell surface modifications were analyzed. The results showed that aged human lymphocytes and freshly isolated rat lymphocytes were more prone to undergo apoptosis. Therefore, the same apoptotic signal is recognized by human and rat lymphocytes, but the extent of the answer is related to the species, to the intensity of the apoptotic stimulus and to the metabolic/developmental condition of the cells. Surface modifications (lipids and glycans), typical of apoptosis, were observed. Our data showed that cell surface changes are species and age dependent. They are early events, progressively achieved in the course of the apoptotic process involving lateral membrane movements of molecules.     

Identification of novel p53-binding proteins by biomolecular interaction analysis combined with tandem mass spectrometry

Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analysis (BIA) to develop a method of direct protein identification after real-time analysis of protein-protein interactions. Using this method, called BIA-MS/MS, we detected multiple p53-interacting proteins in whole tissue extracts from human placenta and liver. Peptide sequencing revealed three proteins whose interaction with p53 had not been previously reported: a cyclin-dependent kinase inhibitor p57/Kip2, a serine/threonine protein phosphatase PP1C, and hemoglobin. Using our system, unambiguous sequence information can be obtained at the femto- to picomole level after repeating the recovery procedure five times. Furthermore, the association and dissociation constants are easily determined by kinetic analysis. This system provides a powerful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner.     

Diving behaviour, dive cycles and aerobic dive limit in the platypus Ornithorhynchus anatinus

We investigated the diving behaviour, the time allocation of the dive cycle and the behavioural aerobic dive limit (ADL) of platypuses (Ornithorhynchus anatinus) living at a sub-alpine Tasmanian lake. Individual platypuses were equipped with combined data logger-transmitter packages measuring dive depth. Mean dive duration was 31.3 s with 72% of all dives lasting between 18 and 40 s. Mean surface duration was 10.1 s. Mean dive depth was 1.28 m with a maximum of 8.77 m. Platypuses performed up to 1600 dives per foraging trip with a mean of 75 dives per hour. ADL was estimated by consideration of post-dive surface intervals vs. dive durations. Only 15% of all dives were found to exceed the estimated ADL of 40 s, indicating mainly aerobic diving in the species. Foraging platypuses followed a model of optimised recovery time, the optimal breathing theory. Total bottom duration or total foraging duration per day is proposed as a useful indicator of foraging efficiency and hence habitat quality in the species.