Conformationally constrained inhibitors of caspase-1 (interleukin-1 beta converting enzyme) and of the human CED-3 homologue caspase-3 (CPP32, apopain)

A systematic study of interleukin-1 beta converting enzyme (ICE, caspase-1) and caspase-3 (CPP32, apopain) inhibitors incorporating a P2-P3 conformationally constrained dipeptide mimetic is reported. Depending on the nature of the P4 substituent, highly selective inhibitors of both Csp-1 or Csp-3 were obtained.     

Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.     

Modulated dispersion explains changes in arrhythmia vulnerability during premature stimulation of the heart

BACKGROUND: Previously, we have shown that a premature stimulus can significantly modulate spatial gradients of ventricular repolarization (ie, modulated dispersion), which result from heterogeneous electrophysiological properties between cells. The role modulated dispersion may play in determining electrical instability in the heart is unknown. METHODS AND RESULTS: To determine if premature stimulus-induced changes in repolarization are a mechanism that governs susceptibility to cardiac arrhythmias, optical action potentials were recorded simultaneously from 128 ventricular sites (1 cm2) in 8 Langendorff-perfused guinea pig hearts. After baseline pacing (S1), a single premature stimulus (S2) was introduced over a range of S1S2 coupling intervals. Arrhythmia vulnerability after each premature stimulus was determined by measurement of a modified ventricular fibrillation threshold (VFT) during the T wave of each S2 beat (ie, S2-VFT). As the S1S2 interval was shortened to an intermediate value, spatial gradients of repolarization and vulnerability to fibrillation decreased by 51+/-9% (mean+/-SEM) and 73+/-45%, respectively, compared with baseline levels. As the S1S2 interval was further shortened, repolarization gradients increased above baseline levels by 54+/-30%, which was paralleled by a corresponding increase (37+/-8%) in vulnerability. CONCLUSIONS: These data demonstrate that modulation of repolarization gradients by a single premature stimulus significantly influences vulnerability to ventricular fibrillation. This may represent a novel mechanism for the formation of arrhythmogenic substrates during premature stimulation of the heart.     

Mechanical debulking versus balloon angioplasty for the treatment of diffuse in-stent restenosis

HeLa cells exposed to cisplatin undergo cell death, presenting morphological and biochemical characteristics typical of apoptosis. In this study we demonstrate that this process is independent of RNA and protein synthesis, since it was not inhibited by actinomycin D or cycloheximide. These substances induced apoptosis by themselves, suggesting an unidentified short-lived inhibitor. The presence of Ca2+ chelators (EDTA and EGTA) did not have effect in this process, excluding the participation of extracellular Ca2+ access. Finally, zinc ions inhibited the low molecular weight DNA degradation and the apoptotic bodies production, but not cell death. These results provide an insight into the mechanism of action of one of the most used antineoplastic drug.     

Prolongation of the p53 response to DNA strand breaks in cells depleted of PARP by antisense RNA expression

The observation that 3-aminobenzamide, which inhibits a variety of ADP-ribose transferases, prolongs the gamma-irradiation-induced increase in intracellular p53 concentration suggested that one or more of such enzymes may determine the duration of the p53 response during G1 arrest. The role of poly(ADP-ribose) polymerase (PARP), an abundant nuclear enzyme activated by DNA strand breaks, in the p53 response to y-irradiation was investigated in Burkitt's lymphoma AG876 cells stably transfected with an inducible PARP antisense construct. Immunoblot analysis revealed that the cellular content of PARP was reduced to virtually undetectable levels after incubation of transfected cells for 72 h with the inducer dexamethasone. In noninduced antisense cells, the p53 concentration reached a maximum 2 h after exposure to 6.3 Gy of gamma-radiation and returned to control values by 4 h. In contrast, the p53 response in PARP-depleted antisense cells peaked at 4 h, with the levels of p53 remaining elevated for up to 12 h after y-irradiation. The maximal increase in p53 concentration was similar in both induced and noninduced cells. These results thus indicate that PARP activity, in part, determines the duration, but not the magnitude, of the p53 response to DNA damage.     

Spectroscopic imaging of the knee with line scan CPMG sequences

OBJECTIVE: A line scan spectroscopic imaging method providing variable T2-weighted spectra from many small voxels along selected tissue columns was applied to study the chemical composition of hematopoietic and fatty marrow in the knees of adults and children. MATERIALS AND METHODS: Line scan Carr-Purcell-Meiboom-Gill (CPMG) spectroscopic imaging sequences were implemented on a 1.5 T clinical scanner. Variable T2-weighted proton spectra from 128 locations along 20 cm long, 5 mm2 columns oriented superiorly to inferiorly through knees were collected from eight healthy adults and eight children. RESULTS: In adult yellow marrow, olefinic protons, water, a composite lipid proton peak, and methyl/methylene protons contributed 6.4 +/- 0.4, 4.2 +/- 1.5, 7.2 +/- 0.5, and 82.2 +/- 1.9% (mean +/- SD) to the spectra, respectively. Marrow spectra were largely independent of position along the column. Marrow spectra of normal children showed distinct positional dependences. Epiphyseal marrow spectra of children (8-16 years old) resembled adult spectra but with more water (mean 15 vs. 4%). Metaphyseal marrow had higher, variable water content, reflecting the extent of marrow conversion and generally obscuring the olefinic proton peak. CONCLUSIONS: Spectroscopic imaging of columns is a time-efficient method for sampling extensive regions of bone marrow with high spatial resolution. It should prove useful for proton spectroscopic studies of hematologic pathologies and conditions requiring the monitoring of lipid composition.