Internucleosomal DNA cleavage in apoptotic WEHI 231 cells is mediated by a chymotrypsin-like protease

Although several lines of evidence support a role for serine proteases in apoptosis, little is known about the mechanisms involved. In the present study, we have examined the apoptosis-inducing potential and dissected the death-signalling pathways of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-tosyl-L-lysine chloromethyl ketone (TLCK), inhibitors of chymotrypsin- and trypsin-like proteases, respectively. Our results designate two distinct roles for serine proteases. Firstly, we show that both inhibitors induce biochemical and morphological characteristics of apoptosis, including proteolysis of poly(ADP-ribose) polymerase 1 (PARP-1) and inhibitor of caspase-activated DNase (ICAD), as well as mitochondrial dysfunction, and that their action is abrogated by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (z-VAD.fmk). These results suggest that inhibition of anti-apoptotic serine proteases governs the onset of the caspase-dependant apoptotic cascade. Secondly, we also demonstrate the involvement of a serine protease in the terminal stage of apoptosis. We showed that chymotrypsin-like protease activity is required for internucleosomal DNA fragmentation in apoptotic cells. Hence, DNA fragmentation is abrogated in TPCK-pre-treated WEHI 231 cells undergoing apoptosis triggered either by anti-IgM or TLCK. These results indicate that internucleosomal DNA cleavage in apoptotic cells is mediated by a chymotrypsin-like protease.     

Correlation of MFOLD-predicted DNA secondary structures with separation patterns obtained by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis

We have studied 57 different mutations within three beta-globin gene promoter fragments with sizes 52 bp, 77 bp, and 193 bp by fluorescent capillary electrophoresis CE-SSCP analysis. For each mutation and wild type, energetically most-favorable predicted secondary structures were calculated for sense and antisense strands using the MFOLD DNA-folding algorithm in order to investigate if any correlation exists between predicted DNA structures and actual CE migration time shifts. The overall CE-SSCP detection rate was 100% for all mutations in three studied DNA fragments. For shorter 52 bp and 77 bp DNA fragments we obtained a positive correlation between the migration time shifts and difference in free energy values of predicted secondary structures at all temperatures. For longer 193 bp beta-globin gene fragments with 46 mutations MFOLD predicted different secondary structures for 89% of mutated strands at 25 degrees C and 40 degrees C. However, the magnitude of the mobility shifts did not necessarily correlate with their secondary structures and free energy values except for the sense strand at 40 degrees C where this correlation was statistically significant (r = 0.312, p = 0.033). Results of this study provided more direct insight into the mechanism of CE-SSCP and showed that MFOLD prediction could be helpful in making decisions about the running temperatures and in prediction of CE-SSCP data patterns, especially for shorter (50-100 bp) DNA fragments.     

TA-clustering: cluster analysis of gene expression profiles through Temporal Abstractions

This paper describes a new technique for clustering short time series of gene expression data. The technique is a generalization of the template-based clustering and is based on a qualitative representation of profiles which are labelled using trend Temporal Abstractions (TAs); clusters are then dynamically identified on the basis of this qualitative representation. Clustering is performed in an efficient way at three different levels of aggregation of qualitative labels, each level corresponding to a distinct degree of qualitative representation. The developed TA-clustering algorithm provides an innovative way to cluster gene profiles. We show the developed method to be robust, efficient and to perform better than the standard hierarchical agglomerative clustering approach when dealing with temporal dislocations of time series. Results of the TA-clustering algorithm can be visualized as a three-level hierarchical tree of qualitative representations and as such easy to interpret. We demonstrate the utility of the proposed algorithm on a set of two simulated data sets and on a study of gene expression data from S. cerevisiae.     

The current availability of neurological in-patient services in post-communist central and eastern European countries

BACKGROUND AND PURPOSE: Limited information can be obtained as to the availability of neurological in-patient services in the former communist countries of Eastern and Central Europe. The objective was to analyse data received directly from representatives of the particular countries. METHODS: The data were collected under the auspices of the 'First European Cooperation Neurology Workshop' held in April 2000, in Trest, Czech Republic. Neurologists from 15 post-communist countries provided information from their respective countries. Linear trends in graphs including the reliability value R(2) were used in the analysis of correlations. RESULTS: Data from 14 countries were assembled and trends were analysed. CONCLUSIONS: Direct relationships were found between: (1) the average department size and the average catchment area (R(2) = 0.1015); (2) the percentage of districts with a neurological in-patient department and the gross national product (GNP) per capita (R(2) = 0.1359); (3) the average neurological department size and the GNP per capita (R(2) = 0.1135), and (4) the average length of treatment and the number of neurological beds/100,000 inhabitants (R(2) = 0.1745). Inverse relationships were found between: (1) the number of neurological beds/100,000 inhabitants and the average hospital catchment area (R(2) = 0.2105), and (2) the number of neurological beds/100,000 inhabitants and the GNP per capita (R(2) = 0.1144).     

Human skin microcirculation after brachial plexus block evaluated by wavelet transform of the laser Doppler flowmetry signal

BACKGROUND: The skin microcirculation may be evaluated noninvasively by laser Doppler flowmetry and iontophoresis with acetylcholine and sodium nitroprusside. Wavelet transform of the perfusion signal shows periodic oscillations of five characteristic frequencies in the interval 0.0095-1.6 Hz. The aim of the current study was to investigate alterations in skin microcirculation induced by brachial plexus block, with emphasis on the periodic oscillations. METHODS: Healthy nonsmokers undergoing hand surgery (n = 13) were anesthetized with brachial plexus block, using bupivacaine, lidocaine, and epinephrine. Skin microcirculation was evaluated by laser Doppler flowmetry and iontophoresis with acetylcholine and sodium nitroprusside before and after brachial plexus block. Wavelet transform of the perfusion signal was performed. As a control group, 10 healthy nonsmokers were included. RESULTS: In the anesthetized arm, skin perfusion after brachial plexus block increased from 19 (12-30) to 24 (14-39) arbitrary units (P < 0.01). A significant increase was also seen in the contralateral arm from 17 (14-32) to 20 (14-42) arbitrary units (P < 0.01). After brachial plexus block, spectral analysis revealed a significant reduction in relative amplitude of the oscillatory components within the 0.0095- to 0.021- (P < 0.001) and 0.021- to 0.052-Hz (P < 0.001) intervals in the anesthetized arm. CONCLUSION: Alterations in skin microcirculation induced by brachial plexus block can be evaluated by wavelet transform of the laser Doppler flowmetry signal. Brachial plexus block reduces the oscillatory components within the 0.0095- to 0.021- and 0.021- to 0.052-Hz intervals of the perfusion signal. These alterations are related to inhibition of sympathetic activity and a possible impairment of endothelial function.     

Arterial dysplasia with ruptured basilar artery aneurysm: Report of a case

An 11 year old girl died unexpectedly as a result of spontaneous subarachnoid hemorrhage originating from a ruptured giant fusiform aneurysm of the basilar artery. The aneurysm had developed as a long standing complication of arterial fibromuscular dysplasia. This discussion describes the light and electron microscopic findings in the aneurysm and the histologic examination of abnormalities in the muscular arteries of the viscera.     

Epiglottic suture for treatment of laryngomalacia

OBJECTIVES: To present a technique for surgical management of laryngomalacia directed against the basic abnormality of the disease. Considering the cause-consequence relations of the abnormalities, we can distinguish two types of laryngomalacia. In the first, the basic abnormality is the pathological shape of the epiglottis: the epiglottis, which normally stands in an upright position, is characteristically excessively folded, restricting the supraglottic space directly as well as indirectly due to the proximity of the aryepiglottic folds that are attached to its lateral edges. In the second type the abnormality is the backward displacement (ptosis) of a normally shaped epiglottis. All other abnormalities are the consequences of these basic abnormalities. We present a new procedure, the epiglottic suture, to correct the pathological shape of the epiglottis. It is a suture placed transversely on the lingual surface of the epiglottis that unfolds the folded epiglottis and shifts apart the adjacent aryepiglottic folds. PATIENTS AND METHODS: Prospective non-randomized study performed on eight severely distressed patients with laryngomalacia at the University Department for Otorhinolaryngology and Cervicofacial Surgery, Ljubljana, Slovenia. RESULTS AND CONCLUSION: The epiglottic suture enabled normal breathing in all treated children without compromising the airway-protection function of the epiglottis. After an average follow up time of 19.12 months (minimum 7 months and maximum 27 months), we have not noticed any complications or deteriorations of breathing.