Vibrational and electronic circular dichroism study of the interactions of cationic porphyrins with (dG-dC)10 and (dA-dT)10

The interactions of two different porphyrins, without axial ligands-5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin-Cu(II) tetrachloride (Cu(II)TMPyP) and with bulky meso substituents-5,10,15,20-tetrakis(N,N,N-trimethylanilinium-4-yl)porphyrin tetrachloride (TMAP), with (dG-dC)10 and (dA-dT)10 were studied by combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopy at different [oligonucleotide]/[porphyrin] ratios, where [oligonucleotide] and [porphyrin] are the concentrations of oligonucleotide per base-pair and porphyrin, respectively. The combination of VCD and ECD spectroscopy enables us to identify the types of interactions, and to specify the sites of interactions: The intercalative binding mode of Cu(II)TMPyP with (dG-dC)(10), which has been well described, was characterized by a new VCD "marker" and it was shown that the interaction of Cu(II)TMPyP with (dA-dT)10 via external binding to the phosphate backbone and major groove binding caused transition from the B to the non-B conformer. TMAP interacted with the major groove of (dG-dC)10, was semi-intercalated into (dA-dT)10, and caused significant variation in the structure of both oligonucleotides at the higher concentration of porphyrin. The spectroscopic techniques used in this study revealed that porphyrin binding with AT sequences caused substantial variation of the DNA structure. It was shown that VCD spectroscopy is an effective tool for the conformational studies of nucleic acid-porphyrin complexes in solution.     

Calcineurin activation is only one calcium-dependent step in cytotoxic T lymphocyte granule exocytosis

We have tested the idea that calcineurin, a calcium-dependent phosphatase that is critical for activating cytokine gene expression in helper T cells, plays a role in lytic granule exocytosis in cytotoxic T lymphocytes (CTLs). We used TALL-104 human leukemic CTLs as a model. Our results confirm an earlier report (Dutz, J. P., Fruman, D. A., Burakoff, S. J., and Bierer, B. E. (1993) J. Immunol. 150, 2591-2598) that immunosuppressive drugs inhibit exocytosis in CTLs stimulated either via the T cell receptor (TCR) or via TCR-independent soluble agents. Of the two recently reported alternate targets of immunosuppressive drugs (Matsuda, S., Shibasaki, F., Takehana, K., Mori, H., Nishida, E., and Koyasu, S. (2000) EMBO Rep. 1, 428-434 and Matsuda, S., and Koyasu, S. (2000) Immunopharmacology 47, 119-125), JNK is not required for lytic granule exocytosis, but we were not able to exclude a role for P38. Exocytosis could be inhibited by expressing GFP fused to a C-terminal fragment of CAIN (cabin 1), but not by expressing VIVIT-GFP. Finally, expressing either full-length or truncated constitutively active mutant calcineurin A enhanced lytic granule exocytosis. However, the mutant calcineurin was unable to support exocytosis when cells were stimulated in the absence of Ca2+ influx. Taken together, our results support the idea that activation of calcineurin is required for lytic granule exocytosis but suggest that it is not the sole Ca2+-dependent step.     

The Implication of Early Chromatin Changes in X Chromosome Inactivation

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Predicting ratings of perceived exertion in youth soccer using decision tree models

The purpose of this study was to determine the effectiveness of white-box decision tree models (DTM) for predicting the rating of perceived exertion (RPE). The second aim was to examine the relationship between RPE and external measures of intensity in youth soccer training at the group and individual level. Training load data from 18 youth soccer players were collected during an in-season competition period. A total of 804 training observations were undertaken, with a total of 43 ± 17 sessions per player (range 12–76). External measures of intensity were determined using a 10 Hz GPS and included total distance (TD, m/min), high-speed running distance (HSR, m/min), PlayerLoad (PL, n/min), impacts (n/min), distance in acceleration/deceleration (TD ACC/TD DEC, m/min) and the number of accelerations/decelerations (ACC/DEC, n/min). Data were analysed with decision tree models. Global and individualized models were constructed. Aggregated importance revealed HSR as the strongest predictor of RPE with relative importance of 0.61. HSR was the most important factor in predicting RPE for half of the players. The prediction error (root mean square error [RMSE] 0.755 ± 0.014) for the individualized models was lower compared to the population model (RMSE 1.621 ± 0.001). The findings demonstrate that individual models should be used for the assessment of players’ response to external load. Furthermore, the study demonstrates that DTM provide straightforward interpretation, with the possibility of visualization. This method can be used to prescribe daily training loads on the basis of predicted, desired player responses (exertion).     

LacSwitchTM Inducible Mammalian Expression System in mouse Swiss 3T3 fibroblasts

Swiss 3T3 fibroblasts were transfected with the provided plasmids of LacSwitch Inducible Mammalian Expression System (Stratagene). Stable transfectants were selected, expanded and characterised. At first, the production of CAT in these cell lines could be induced by IPTG treatment, but the inducibility was lost after a few months in culture in a reproducible manner. Further analysis revealed that the transfectants did not lose the cat gene nor the lac repressor protein. As a result, we conclude that LacSwitch Inducible Mammalian Expression System needs further modification for use in Swiss 3T3 fibroblasts.     

Identification of the tautomeric form of formycin A in its complex with<Emphasis Type="Italic"> Escherichia coli</Emphasis> purine nucleoside phosphorylase based on the effect of enzyme–ligand binding on fluorescence and phosphorescence

Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, formycin A (FA), a close structural analogue of adenosine (natural substrate), in the absence and presence of phosphate (P_i, substrate). Formation of enzyme–FA complexes led to marked quenching of enzyme tyrosine intrinsic fluorescence and phosphorescence, with concomitant increases in fluorescence and phosphorescence of FA. Fluorescence resonance energy transfer from the protein Tyr160 residue to the FA base moiety was identified as a major mechanism of protein fluorescence quenching, increased by addition of P_i. The effects of enzyme–FA interactions on the nucleoside excitation and emission spectra for fluorescence and phosphorescence revealed shifts in the tautomeric equilibrium of the bound FA, i.e. from the N(1)–H tautomer (predominant in solution) to the N(2)–H form, enhanced by the presence of P_i. The latter was confirmed by enzyme–ligand dissociation constant (K _d) values of 5.9±0.4 and 2.1±0.3 M in the absence and presence of P_i, respectively. Addition of glycerol (80%, v/v) led to a lower enzyme affinity (K _d70 M), without changes in binding stoichiometry. Enzyme–FA complex formation led to a higher increase of the fluorescence than the phosphorescence band of the ligand, consistent with the fact that the N(2)–H tautomer is characterized by a weaker phosphorescence than the N(1)–H tautomeric form. These results show, for the first time, the application of phosphorescence spectroscopy to the identification of the tautomeric form of the inhibitor bound by the enzyme.Abbreviations Ado adenosine - FA formycin A [3-(-d-ribofuranosyl)-7-aminopyrazolo[4,3-d]pyrimidine] - FB formycin B - FRET fluorescence resonance energy transfer - Guo guanosine - Hepes N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - Ino inosine - m¹FA N(1)-methylformycin A - m²FA N(2)-methylformycin A - m⁴FA N(4)-methylformycin A - m⁶FA N(6)-methylformycin A - m⁷Guo N(7)-methylguanosine - P_i orthophosphate - PNP purine nucleoside phosphorylase - Xao xanthosine     

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.