Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcineurin binding and cellular location.

The immunosuppressants cyclosporin A (CsA) and FK506 appear to block T-cell function by inhibiting the calcium-regulated phosphatase calcineurin. While multiple distinct intracellular receptors for these drugs (cyclophilins and FKBPs, collectively immunophilins) have been characterized, the functionally active ones have not been discerned. We found that overexpression of cyclophilin A or B or FKBP12 increased T-cell sensitivity to CsA or FK506, respectively, demonstrating that they are able to mediate the inhibitory effects of their respective immunosuppressants in vivo. In contrast, cyclophilin C, FKBP13, and FKBP25 had no effect. Direct comparison of the Ki of each drug-immunophilin complex for calcineurin in vitro revealed that although calcineurin binding was clearly necessary, it was not sufficient to explain the in vivo activity of the immunophilin. Subcellular localization was shown also to play a role, since gene deletions of cyclophilins B and C which changed their intracellular locations altered their activities significantly. Cyclophilin B has been shown previously to be located within calcium-containing intracellular vesicles; its ability to mediate CsA inhibition implies that certain components of the signal transduction machinery are also spatially restricted within the cell.     

Chromatin very small angle neutron scattering: further evidence for a 30 nm diameter super coil in dilute solutions.

Intact chromatin, chromatin minus histone H1, and nuclease digestion fragments have been studied by very small angle neutron scattering. The results are not consistent with a straight chain of nucleosomes and require the presence of a higher order coiling in monovalent salt solutions. The data are interpretable by a structure having a cross section radius of gryration of 8.5 +/- 1 nm, which suggests an outer diameter for a coil of nucleosomes of 27 +/- 3 nm.     

Chromatin fiber dimensions and nucleosome orientation: a neutron scattering investigation.

Chromatin fibers were studied in solutions of mM monovalent salt by small angle neutron scattering. The variation of the cross section radius of gyration with H2O/D2O contrast shows that DNA is at much larger average radial distances from the fiber axis than histone. Consequently, the coils of DNA in a core particle must be approximately parallel to the fiber direction. The radii of gyration suggest that the maximum diameter of chromatin and nucleosomes is approximately 14 nm and that the DNA id distributed in two radial layers. The concentration dependence of the scattering maxima near 14 nm spacings furnishes independent support for a 14 nm external diameter and can be interpreted by a double DNA layer configuration.     

Quantifying BSE control by calculating the basic reproduction ratio R 0 for the infection among cattle

The safety of using meat and bone meal (MBM) in mammal feed was studied in view of BSE, by quantifying the risk of BSE transmission through different infection routes. This risk is embodied in the basic reproduction ratio R ₀ of the infection, i.e. the average number of new infections induced by one initial infection. Only when R ₀ is below 1, will the disease die out with certainty and the population will become free from BSE. Unfortunately this is a slow process due to the slow progression of the disease. We calculate R ₀ explicitly from basic ingredients taking several different transmission routes into account. Several of the basic ingredients are functions of age or of infection-age. We also calculate the exponential growth rate r in terms of the same basic ingredients. Next we quantify the ingredients from available data and compute the effects on R ₀ of various scenarios for controlling BSE, with examples for the UK and the Netherlands. This revised version was published online in November 2003. A correction was made to formula 10 of this paper, indicies were previously printed in an incorrect order and an extraneous element has been removed.     

DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway

Type II restriction endonucleases protect bacteria against phage infections by cleaving recognition sites on foreign double-stranded DNA (dsDNA) with extraordinary specificity. This capability arises primarily from large conformational changes in enzyme and/or DNA upon target sequence recognition. In order to elucidate the connection between the mechanics and the chemistry of DNA recognition and cleavage, we used a single-molecule approach to measure rate changes in the reaction pathway of EcoRV and BamHI as a function of DNA tension. We show that the induced-fit rate of EcoRV is strongly reduced by such tension. In contrast, BamHI is found to be insensitive, providing evidence that both substrate binding and hydrolysis are not influenced by this force. Based on these results, we propose a mechanochemical model of induced-fit reactions on DNA, allowing determination of induced-fit rates and DNA bend angles. Finally, for both enzymes a strongly decreased association rate is obtained on stretched DNA, presumably due to the absence of intradomain dissociation/re-association between non-specific sites (jumping). The obtained results should apply to many other DNA-associated proteins.     

Determination of the Selection Capacity of Antibiotics for Gene Selection

Choosing a potent selection antibiotic (SA), is a crucial success factor when creating stably transfected cell lines using an antibiotic selection marker. The selection capacity of this antibiotic is defined as its ability to kill sensitive, untransfected parental cells, while leaving resistant, transfected cells unharmed. Currently, no procedure has been described to determine this selection capacity. Therefore, a protocol to obtain a numerical value, called the “selectivity factor” (SF), that defines the selection capacity of SAs is developed. The SF is determined by using a modified MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐diphenyltetrazolium bromide) assay for both sensitive and resistant cells, and applies to commonly used cell lines. To prove the concept, the SF of the SA G418 and hygromycin B (HmB) on several cell lines is determined. The SF of G418 on BHK‐21 cells is very high, indicating that G418 is an ideal SA for transfected BHK‐21 cells. For HeLa cells, the SF of G418 is very low suggesting G418 is not an optimal SA for selecting transfected HeLa cells. For these cells, HmB would be a better choice. These conclusions are confirmed by an independent cell death assay. The SF identifies the most optimal SA for a certain cell line, reduces the risk of selecting spontaneously resistant cell clones, and streamlines the process of generating stable cell lines. Most importantly, the method is especially time saving when obtaining stable cell lines expressing toxic genes, and reduces culture times for generating large numbers of cell lines from the same parental cell line.     

A new technique of coronary artery ligation: Experimental myocardial infarction in rats in vivo with reduced mortality

In vivo models of myocardial infarction following coronary artery ligation in the rat still suffer from high early mortality and a low rate of success of myocardial infarction. This study investigated the possibility of reducing early mortality and increasing the rate of myocardial infarction by modifications of surgical techniques. Eighteen rats were divided into two groups: normal control (3 rats) and ligation (15 rats). The major modifications of surgical techniques used in this study include: (1) no exteriorization of the heart, (2) ligation of the origins of the branches rather than the main trunk of the left coronary artery, (3) removal of air from the chest after closure, (4) supplying oxygen immediately after extubation. Following surgery, the rats recovered uneventfully and 11 rats were alive after 16 weeks. One rat, with a large myocardial infarction, died 2 h after surgery. Early mortality (during surgery and 1 week after surgery) was 6.7% with a success rate of myocardial infarction of 85%. The left ventricle in the ligation group showed significant dilation relative to normal and shamoperated control hearts (317% of control hearts, p < 0.001). However, myocardial mass did not increase. The average infarct size was 33%. These results demonstrate that a reduction in early mortality and an increased success rate of myocardial infarction can be achieved by modifications of surgical techniques.