Photochemical mechanism of DEACM uncaging: a combined time-resolved spectroscopic and computational study.

The elementary steps of photocleavage in (coumarin-4-yl)methyl photoremovable protecting groups (PPGs) are elucidated by a combined electronic structure and time-resolved visible pump infrared probe (VIS-pump IR-probe) spectroscopic study. We specifically focus on the [7-(diethylamino)coumarin-4-yl]methyl (DEACM) PPG which has found increasing interest in biological applications over recent years. A series of leaving groups (LGs) are investigated, including azide (DEACM-N3), thiocyanate (DEACM-SCN), carbonate (DEACM-Carb), and a thymine nucleobase (DEACM-T) representing a model system for caged DNA. These systems are found to exhibit vastly different photocleavage time scales, ranging from the subpicosecond scale in the case of DEACM-SCN to ∼35 picoseconds in the case of DEACM-N3 and ∼540 picoseconds in the case of DEACM-Carb. In the case of DEACM-SCN, the appearance of the product is biphasic, with a fast (

A light-triggered transmembrane porin.

Porins are ideal model systems for channel engineering. OmpG is a robust, monomeric, transmembrane ß-barrel without ion selectivity. Here, we present a photocaged diethylaminocoumarin (DEACM) hybrid of OmpG. Blockage of the pore by DEACM is confirmed by reduced conductivity. An optimal effect was obtained when two bulky butyl-substituted coumarin cages were attached on the inside of the pore. Irradiation at 385 nm removed the photocages, leading to a restoration of channel conductivity.

Picosecond activation of the DEACM photocage unravelled by VIS-pump-IR-probe spectroscopy.

The light-induced ultrafast uncaging process of the [7-(diethylamino)coumarin-4-yl]methyl (DEACM) cage is measured by time-resolved visible-pump-infrared-probe spectroscopy, and supported by steady-state absorption spectroscopy in the visible and infrared spectral regions. Understanding the uncaging process is important because its favorable properties make DEACM an interesting case for chemical and biological applications. It has a convenient absorption in the visible spectral range, and is relatively easily modified to carry leaving groups (LGs) such as nucleotides, substrates or inhibitors, which are inactive when bound and active when released. Previous work suggested a lower limit for the uncaging rate, which places it among the fastest available cages. Here, we determine the photodissociation directly to occur on the picosecond time scale by monitoring the appearance of the released LG in the infrared spectral region. In the present study, azide (N3) is chosen as an LG to monitor photodissociation because its vibrational mode is spectrally isolated (hence easy to follow) and its absorption wavenumber is sensitive to local structural rearrangements. The uncaging process is recorded up to 3 nanoseconds and compared to the collected steady-state spectra. The free LG appears on a picosecond time scale, rendering this one of the fastest known cages. No evidence is found for a tight-ion pair (TIP) preceding the free LG. The uncaging mechanism is found to be slowed down upon the addition of water to acetonitrile.

Peripheral Nerve Diffusion Tensor Imaging: Assessment of Axon and Myelin Sheath Integrity.

PURPOSE: To investigate the potential of diffusion tensor imaging (DTI) parameters as in-vivo biomarkers of axon and myelin sheath integrity of the median nerve in the carpal tunnel as validated by correlation with electrophysiology. METHODS: MRI examinations at 3T including DTI were conducted on wrists in 30 healthy subjects. After manual segmentation of the median nerve quantitative analysis of fractional anisotropy (FA) as well as axial, radial and mean diffusivity (AD, RD, and MD) was carried out. Pairwise Pearson correlations with electrophysiological parameters comprising sensory nerve action potential (SNAP) and compound muscle action potential (CMAP) as markers of axon integrity, and distal motor latency (dml) and sensory nerve conduction velocity (sNCV) as markers of myelin sheath integrity were computed. The significance criterion was set at P=0.05, Bonferroni corrected for multiple comparisons. RESULTS: DTI parameters showed a distinct proximal-to-distal profile with FA, MD, and RD extrema coinciding in the center of the carpal tunnel. AD correlated with CMAP (r=0.50, p=0.04, Bonf. corr.) but not with markers of myelin sheath integrity. RD correlated with sNCV (r=-0.53, p=0.02, Bonf. corr.) but not with markers of axon integrity. FA correlated with dml (r=-0.63, p=0.002, Bonf. corr.) and sNCV (r=0.68, p=0.001, Bonf. corr.) but not with markers of axon integrity. CONCLUSION: AD reflects axon integrity, while RD (and FA) reflect myelin sheath integrity as validated by correlation with electrophysiology. DTI parameters consistently indicate a slight decrease of structural integrity in the carpal tunnel as a physiological site of median nerve entrapment. DTI is particularly sensitive, since these findings are observed in healthy participants. Our results encourage future studies to evaluate the potential of DTI in differentiating axon from myelin sheath injury in patients with manifest peripheral neuropathies.

Rapid method for determination of the activity concentrations of (89)Sr and (90)Sr.

This paper describes a rapid method for determining the activity concentrations of (89)Sr and (90)Sr in air and water using LSC. After sample preparation strontium is separated by extraction chromatography. The chemical yield is determined by X-ray fluorescence. The spectra of the Cerenkov radiation and beta radiation are taken with a LSC. From both spectra the activities of strontium are calculated by means of a spectrum deconvolution method. The results can be obtained within 4.5 hours for particulate filters and 2 hours for water.

Heterotopic expression of the Xl-Fli transcription factor during Xenopus embryogenesis: modification of cell adhesion and engagement in the apoptotic pathway.

In the Xenopus laevis embryo, the overexpression of the Xl-FLI protein, a transcription factor of the ETS family, provokes severe developmental anomalies, which affect anteroposterior and dorsoventral polarities, optic cup formation, head cartilage morphogenesis, and erythrocyte differentiation. It has been proposed that these effects could be correlated to modifications of cell adhesion properties and/or to an increased engagement of cells in the apoptotic pathway during early development (Remy et al., Int. J. Dev. Biol. 40, 577-589, 1996). To address these questions, we have first analyzed the behavior of cells overexpressing the protein in both aggregation and adhesion assays. We observe perturbations of cell-cell interactions as well as perturbations of cell adhesion and spreading on fibronectin and extracellular matrix (ECM). Second, we have analyzed apoptosis of cells overexpressing the Xl-FLI protein, by testing DNA fragmentation, caspase-3 activity and by performing TUNEL assay. We show that Xl-Fli overexpression results in the appearance of hallmarks of apoptosis, including exclusion of cells from the interior of the embryo, internucleosomal fragmentation of DNA and dose-dependent induction of caspase-3, resulting in the hydrolysis of poly(ADP-ribose) polymerase. In addition, a dominant-negative mutation of BMPs receptors decreases the effects of Xl-Fli overexpression, suggesting that a modification of the BMP signalling could be responsible for increased apoptosis. The latter appears to affect predominantly ventral and ventrolateral regions of the embryo.

Immobilization of TADDOL with a High Degree of Loading on Porous Silica Gel and First Applications in Enantioselective Catalysis.

The versatile TADDOL moiety has been immobilized for the first time on an inorganic support (highly porous silica gel). This gives access to Ti - Lewis acids (see picture), which turn out to be very efficient in two standard enantioselective reactions. X=OiPr, OTos.

Xl erg: expression pattern and overexpression during development plead for a role in endothelial cell differentiation.

The ets gene family encodes transcription factors related to the proto-oncogene c-ets-1 and involved in cell proliferation, differentiation, and oncogenic transformation. We have characterized the Xenopus homologue of the human erg gene, an ets-related-gene, and its expression has been examined throughout early embryonic development. Xl erg encodes at least two proteins, resulting from alternative splicing events. The transcripts are restricted to the forming endocardium, the endothelial cells of the blood vessels and to the neural crest-derived mesenchyme cells of the pharyngial arches. When Xl ERG is expressed ectopically in Xenopus embryos by microinjection of synthetic mRNA, multiple developmental defects are observed. Dorsally injected embryos have their AP axis shortened and present severe defects in eye and somite morphogenesis. Ventrally injected embryos show a posteriorization of the cells having received the message together with ectopic endothelial cell differentiation as revealed by the accumulation of X-msr transcripts. In both cases, accumulation of erythrocytes in structures not connected with the blood circulatory system can be observed. Our data suggest that Xl erg may be involved in cell motility and in the development of the circulatory system.

BIBW22 BS, potent multidrug resistance-reversing agent, binds directly to P-glycoprotein and accumulates in drug-resistant cells.

The expression of P-glycoprotein (P-gp) in tumor cells causes a multidrug resistance (MDR) phenotype. P-gp has been shown to mediate the transport of structurally dissimilar drugs across the cell membrane in an energy-dependent manner. In this report, we show that BIBW22 BS, a phenylpteridine analog, reverses the MDR phenotype of CEM human lymphoma cells in a dose-dependent fashion. Using a photoactive analog of BIBW22 BS {[3H]azido-4-[N-(2-hydroxy-2-methylpropyl)-ethanolamino]-2, 7-bis(cis-2,6-dimethyl-morpholino)-6-phenylpteridine}, we show the photoaffinity labeling of a 170-kDa protein in drug-resistant cells immunoprecipitated with P-gp-specific monoclonal antibodies. The photolabeling of P-gp by [3H]azido-BIBW22 BS was specific and saturable. Furthermore, BIBW22 BS, vinblastine, and verapamil, but not colchicine, inhibited the photolabeling of P-gp by [3H]azido-BIBW22 BS. Drug binding studies showed that membranes from MDR cells bound more BIBW22 BS than parental drug-sensitive cells, and this binding was inhibited with vinblastine and, to a lesser extent, with uridine. However, drug transport studies demonstrated that BIBW22 BS is not a substrate for P-gp efflux pump. Interestingly, BIBW22 BS was shown to accumulate more in resistant cells. Also, BIBW22 BS accumulation in drug-sensitive and -resistant cells was not energy dependent. These results are in contrast with the observed decrease in accumulation or enhanced efflux of [3H]vinblastine seen in the same MDR cells. A comparison of [3H]azido-BIBW22 BS or [3H]azidopine photolabeled P-gp by Cleveland mapping with Staphylococcus aureus V8 protease showed differences in the photolabeled peptides. Taken together, the results of this study show that BIBW22 BS is a potent MDR-reversing agent that binds directly to P-gp but is not effluxed from drug-resistant cells.

Modulation of multidrug resistance by BIBW22BS in blasts of de novo or relapsed or persistent acute myeloid leukemia ex vivo.

The phenylpteridine derivative BIBW22BS (BIBW22) is a potent modulator of multidrug resistance (MDR). We investigated BIBW22 in comparison to dexniguldipine and verapamil as modifier of MDR in blasts of de novo, relapsed or persistent acute myeloid leukemia (AML) in vitro. All patients with relapsed or persistent AML had been pretreated with idarubicin and cytosine arabinoside. The degree of MDR was determined by efflux kinetics of rhodamine 123 (R123), daunorubicin, and idarubicin measured by flow cytometry (FACS). A total of 51 patients with AML, 25 de novo and 26 relapsed or persistent, were investigated. While only 6 out of 25 de novo AML blast populations showed moderate efflux of R123 and daunorubicin, 17 out of 26 blast populations of relapsed or persistent AML had an efflux between 20% and 44% within 15 min ex vivo. This efflux could be significantly inhibited by 1 microM BIBW22, 1 microM dexniguldipine, or 10 microM verapamil. For idarubicin we found an effusion of 40+/-9% within 15 min in all blast populations that could not be inhibited by the modulators. Clinically achievable drug concentrations causing only moderate side-effects are in the range of 0.5 microM dexniguldipine and 3 microM verapamil. Up to now, BIBW22 has not been investigated clinically. Thus the potential toxicity of concentrations of 0.5-1 microM BIBW22, sufficient for an optimal efflux inhibition ex vivo, is not known yet. We conclude from our ex vivo investigations in blast populations of de novo, relapsed or persistent AML that BIBW22 is a potent modulator of MDR.

Biochemical modulation of 'classical' multidrug resistance by BIBW22BS, a potent derivative of dipyridamole.

BACKGROUND: Modulators of the 'classical' multidrug resistance (mdr) phenotype have low efficacy in patients with solid tumors. We analyzed BIBW22BS, 4-[N-(2-hydroxy-2-met- hyl-propyl)-ethanolamino]-2,7-bis(cis-2,6-dimethyl-morpho- lino)-6-phenylpteridine, a derivative of dipyridamole, for its higher potential to modulate mdr. MATERIALS AND METHODS: Four human malignant cell lines: BRO, A2780, GLC4, SW1573, the Pgp-positive sublines: BRO/mdr1.1, 2780AD and the non-Pgp sublines: GLC4/ADR, SW1573/2R120 were used in vitro to investigate BIBW22BS as a modulator of the antiproliferative effects of vincristine and doxorubicin and to compare the potency of BIBW22BS with that of dipyridamole, verapamil, bepridil and flunarizine. BRO/mdr1.1 s.c. well-established xenografts in nude mice were used to study the modulating properties of BIBW22BS 50 mg/kg i.v. followed after one h by vincristine 1 mg/kg i.p. or doxorubicin 8 mg/kg i.p. weekly x 2. RESULTS: BIBW22BS was 20- to 100-fold more potent than dipyridamole in the reversal of resistance in the Pgp-positive sublines. Reversal of resistance was obtained in a dose-dependent manner and was complete at concentrations of 0.5-2.5 microM. At non-toxic, equimolar concentrations of 1.0 microM BIBW22BS showed higher modulating potency than the calcium-channel blockers. BIBW22BS did not affect resistance in the non-Pgp sublines. BRO/mdr1.1 s.c. xenografts have stable multidrug-resistance characteristics upon serial transplantation. BIBW22BS, vincristine, or doxorubicin as single agents were not effective in vivo, while the addition of BIBW22BS could significantly reduce the tumor growth expressed as the T/C% of vincristine from 109% to 48% and that of doxorubicin from 55% to 32%. However, reversal of vincristine resistance in BRO/mdr1.1 xenografts was not complete when compared to the efficacy of vincristine in BRO xenografts. CONCLUSION: The results encourage the further preclinical development of BIBW22BS as a modulator of 'classical' multidrug resistance in cancer patients.

6,6-Disubstituted Hex-5-enoic acid derivatives as combined thromboxane A2 receptor antagonists and synthetase inhibitors.

A series of omega-disubstituted alkenoic acid derivatives were designed and synthesized as antithrombotic inhibitors of thromboxane A2 synthetase and thromboxane A2 receptor antagonists. Hexenoic acid derivatives with a 3-pyridyl group and a 4-(2-benzenesulfonamidoethyl)phenyl substituent were found to be optimal with regard to the dual mode of action. The most potent compound, (E)-6-(4-(2-(((4-chlorophenyl)sulfonyl)amino)ethyl)phenyl)-6-(3-pyridyl) hex-5-enoic acid (36), inhibits thromboxane A2 synthetase in gel-filtered human platelets with an IC50 value of 4.5 +/- 0.5 nM (n = 4), whereas an inhibitory effect on cyclooxygenase is seen only at a much higher concentration (IC50: 240 microM). Radioligand-binding studies with [3H]SQ 29,548 in washed human platelets revealed that 36 blocks the prostaglandin H2/thromboxane A2 receptor with an IC50 of 19 +/- 5 nM (n = 5) and is therefore 85-fold more potent than another combined thromboxane A2 synthetase inhibitor/receptor antagonist, Ridogrel (4). Compound 36 inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an EC50 of 1 microM (Ridogrel: 16 microM) and 100 nM, respectively, and was selected for further development.

BIBW 22, a dipyridamole analogue, acts as a bifunctional modulator on tumor cells by influencing both P-glycoprotein and nucleoside transport.

BIBW 22, a phenylpteridine analogue of dipyridamole (DPM), enhanced vincristine cytotoxicity approximately 10 times more than DPM in a multidrug-resistant (MDR) KB V20C cell line. Using rhodamine 123 accumulation in KB V20C cells as an indicator of MDR phenotype, BIBW 22 was shown to be about 100 times more potent than DPM in inhibiting the MDR-associated efflux of rhodamine 123. Photolabeling of P-glycoprotein in KB V20C plasma membranes with 0.2 microM [3H]azidopine was strongly inhibited by 1 microM BIBW 22, indicating that this compound reverses the MDR phenotype by interfering with MDR-associated P-glycoprotein. In addition, BIBW 22 at 1 microM could also enhance the cytotoxicity of 5-fluorouracil in KB cells about 20-fold. Its potency in inhibiting nucleoside transport is 7-fold more potent than that of DPM. These results suggest that BIBW 22 is a potent bifunctional modulator which influences both P-glycoprotein and nucleoside transport in tumor cells. Potential use of this compound as a modulator of combination chemotherapy involving antimetabolites and drugs affected by MDR should be explored.

Energetics of the structure and chain tilting of antiparallel beta-barrels in proteins.

The preferred structural pattern of antiparallel beta-barrels in proteins, described as the right-handed tilting of the peptide strands with respect to the axis of the barrel, is accounted for in terms of intra- and interchain interaction energies. It is related to the preference of beta-sheets for right-handed twisting. Conformational energy computations have been carried out on three eight-stranded antiparallel beta-barrels composed of six-residue strands, in which L-Val and Gly alternate, and having a right-handed, a left-handed, or no tilt. After energy minimization, the relative energies of these structures were 0.0, 8.6, and 46.1 kcal/mol, respectively; i.e., the right-tilted beta-barrel is favored energetically, in agreement with anti-parallel beta-barrels observed in proteins. Tilting of the barrel is favored, relative to the nontilted structure, by both intra- and interstrand interactions, because tilting allows better packing of the bulky side chains. On the other hand, the energy difference between the left- and right-tilted barrels arises essentially from intrachain interactions. This is a consequence of the preference of beta-sheets for a right-handed twist. Space limitations inside the barrel are satisfied if there is an alternation of bulky residues and residues with small or no side chain (preferably Gly) in neighboring positions on adjacent strands. Such a pattern is seen frequently in antiparallel beta-barrels of globular proteins. The computations indicate that a structure with Val...Gly pairs can be accommodated in a beta-barrel with no distortion.

Prediction of the three-dimensional structure of the enzymatic domain of t-PA.

Tissue plasminogen activator (t-PA), an enzyme of the fibrinolytic system, is responsible for lysis of fibrin via activation of plasminogen, and therefore for degradation of blood clots. There are currently no X-ray crystal structure data of the t-PA molecule available either in whole or in part. We therefore predicted the three-dimensional structure of the protease domain by means of computer-graphical methods*. The model obtained forms a basis for understanding the binding of plasminogen to the active site of t-PA. In addition, the interactions of various inhibitors with t-PA were studied by modeling them into the active site. The model also yields an explanation for the observed amidolytic activity of t-PA in the single chain form.

An autoradiographic analysis of the mode of proliferation in the buccal mucosa of rats incubated up to 5 hours.

This study was concerned with the course of DNA synthetic activity (3H-TdR-LI-method) in the buccal mucosa of adult male. Sprague Dawley rats over an incubation period of 5 h. Interest was focussed on the influences of different media and, in particular, on temoporary changes in the proliferative activity. 3H-LI were compared in specimens (a) kept in active (= i.e., 3H-thymidine containing) medium throughout their respective incubation period and (b) pre-incubated in inactive medium for varying, but clearly defined periods before being transferred into active medium for 30 min (actual 3H-LI). Independent of the medium used the rate of DNA synthesis was markedly lowered at 60 min, the reduction being more or less significant in the different media. This was due to a temporary inhibition of DNA synthesis, which was restored after 120-180 min. In contrast, the blockade of G2-phase and/or mitosis persisted up to the end of incubation, as indicated by the unchanged number of labelled mitotic figures after 120 min. Addition of glutamine to Mc Coy's 5A markedly enhanced the activity of 3H-thymidine incorporation, but could not prevent the temporary inhibition of DNA synthesis. The biochemical mechanisms relevant for cell proliferation have been reviewed and correlated to the present results.