Cysteine cathepsins trigger caspase-dependent cell death through cleavage of bid and antiapoptotic Bcl-2 homologues

As a model for defining the role of lysosomal cathepsins in apoptosis, we characterized the action of the lysosomotropic agent LeuLeuOMe using distinct cellular models. LeuLeuOMe induces lysosomal membrane permeabilization, resulting in release of lysosomal cathepsins that cleave the proapoptotic Bcl-2 family member Bid and degrade the antiapoptotic member Bcl-2, Bcl-xL, or Mcl-1. The papain-like cysteine protease inhibitor E-64d largely prevented apoptosis, Bid cleavage, and Bcl-2/Bcl-xL/Mcl-1 degradation. The pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethyl ketone failed to prevent Bid cleavage and degradation of anti-apoptotic Bcl-2 homologues but substantially decreased cell death, suggesting that cathepsin-mediated apoptosis in these cellular models mostly follows a caspase-dependent pathway. Moreover, in vitro experiments showed that one or more of the cysteine cathepsins B, L, S, K, and H could cleave Bcl-2, Bcl-xL, Mcl-1, Bak, and BimEL, whereas no Bax cleavage was observed. On the basis of inhibitor studies, we demonstrate that lysosomal disruption triggered by LeuLeuOMe occurs before mitochondrial damage. We propose that degradation of anti-apoptotic Bcl-2 family members by lysosomal cathepsins synergizes with cathepsin-mediated activation of Bid to trigger a mitochondrial pathway to apoptosis. Moreover, XIAP (X-chromosome-linked inhibitor of apoptosis) was also found to be a target of cysteine cathepsins, suggesting that cathepsins can mediate caspase-dependent apoptosis also downstream of mitochondria.     

Densitometric analysis of dental implant placement between flapless technique and the two-stage technique--a pilot study

Flapless technique is a surgical approach of implant placement without raising a mucoperiosteal flap. Such approach has many advantages: shorter surgical treatment, minimal bleeding, postoperative discomfort for the patient is reduced; possibility of immediate loading of the inserted implant, faster procedure of implant placement and by that less time is needed for the complete implant-prosthetic restoration. Purpose of this pilot study was radiographic assessment of flapless technique and determination of its clinical values in comparison with two-stage dental implant technique through computerized densitometric analysis. The sample consisted of 10 patients with missing teeth in the premolar region in the upper jaw. An implant was placed in that position. In the first group of 5 patients the implants were inserted with the flapless technique, and in the other group of 5 patients implant insertion was done with a two-stage technique. All inserted implants were loaded with metal-ceramic crowns 3 months after placement. The patients were followed for 18 months through clinical follow-ups and radiovisiographical (RVG) images made after 3, 12 and 18 months. After comparing the average densities, the results showed similar decrease of density in both groups, conventional two-stage technique showed 3.24 and flapless technique 1.23. It can be concluded that flapless technique in everyday clinical usage has the same result as the two-stage dental implant technique.     

Foetal Leydig cells and the neuroendocrine system

It has been shown that adult human Leydig cells express a number of neuroendocrine markers, and, therefore, could be considered as a part of the neuroendocrine system in the adult. A limited number of studies have dealt with the dynamics of development of human foetal Leydig cells, whereas studies on their neuroendocrine nature are still extremely rare. Therefore, the aim of our study was to investigate the development of human foetal Leydig cells in different weeks of gestation (wg) and to check if these cells express certain markers characteristic of the diffuse neuroendocrine system (DNS). Qualitative, quantitative histological studies and immunohistochemical analyses of human foetal testicular tissue have been performed. According to our data, Leydig cells formed a dynamic population of cells within the interstitum of testes in the period between 15 and 36 wg. The total number of Leydig cells of human foetal testes changed through different stages of gestation by means of 'pulsatile' dynamics (most likely, by following the variable level of gonadotropins). At early stages of development (15-17 wg) immunohistochemical reactions for the expression of neuron specific enolase (NSE) were positive within sex cords and between them, in the interstitum. Pro-spermatogonia in the sex cords were positive, as well as elongated spindle-shaped cells of the interstitum (very likely, precursors of Leydig cells). During the later stages of development (28-36 wg), excluding the pro-spermatogonia, the interstitial Leydig cells were also positive. The results of the immunohistochemical analyses (the expression of NSE) confirmed the hypothesis that human foetal Leydig cells were of neuroendocrine nature.     

Estimates of forest floor litter frog communities: A comparison of two methods

Estimates of forest leaf litter frog density, mass, richness and diversity given by the widely used 8 m × 8 m large plot method (LPM) were compared with estimates obtained by a newly proposed method (small 2 m × 1 m plots with leaf removal; SPLR). The study site was an undisturbed area of the Atlantic Rainforest of Ilha Grande, an island located in the south of Rio de Janeiro State, Brazil. Twenty‐four LPM (totalling 1536 m² of forest floor) and 90 SPLR (totalling 180 m² of forest floor) were performed. The estimates obtained by the two methods differed markedly, indicating that even using a much smaller sampling area (11.7% of that of LPM), SPLR gave frog density estimates six times higher, and frog mass estimates approximately 2.5 times higher than estimates provided by LPM. The species richness and diversity obtained by the two methods were similar, despite the fact that the total area sampled with SPLR was much smaller. These data suggest that LPM may underestimate the abundance and biomass of leaf litter frogs in a given area.     

The photodynamic activity of 131-[2′-(2-pyridyl)ethylamine] chlorin e6 photosensitizer in human esophageal cancer

A novel 13¹-pyridine substituted chlorin e₆ derivative (Chlorin A) was synthesized. It has characteristic long wavelength absorption at 664?nm and the emission wavelength at 667?nm. The generation rate of singlet oxygen of this compound is higher than Temoporfin. In vitro, Chlorin A showed higher phototoxicity against the human esophageal cancer cells than Temoporfin while with lower dark-toxicity. Its accumulation effect in mitochondria, lysosomes and endoplasmic reticulum was traced in subcellular localization tests. In flow cytometry obvious apoptosis cells were observed after 2?h irradiation. Significant in vivo photodynamic anti-tumor efficacy was also exhibited on mice bearing esophageal cancer. So Chlorin A could be suggested as a promising anti-tumor drug candidate in photodynamic therapy.     

Demonstrability of some oxidative enzymes in early rodent embryos with and without fixation

Several oxidative enzymes [NADH-TR (reduced nicotinamide-adenine dinucleotide-tetrazolium reductase), NADPH-TR (reduced nicotinamide-adenine dinucleotide phosphate-tetrazolium reductase), SDH (succinic dehydrogenase) and LDH (lactate dehydrogenase)] were studied by histochemical means during early development of rat and mouse. All investigated enzymes could be easily demonstrated in zygote and also to some extent in somitic stages without any pretreatment. However, in cleavage and early postimplantation stages enzyme activity could be revealed only after the embryos were pretreated in some way. This pretreatment can be fixation with formalin or acetone, freezing and thawing, slight mechanical damage or very prolonged incubation time. The formazan granules as a sign of enzymatic activity were present in all stages of embryonic development and were more abundant in reactions for NADH-TR and LDH than in reactions for NADPH-TR and SDH. Our results suggest that the investigated enzymes are present in all embryonic cells during early development. It seems that the permeability of embryonic cells for histochemical media must be increased otherwise the histochemical reactions cannot be accomplished.     

Vitalfärbungsversuche mit reduziertem Neutralrot an „vollen“ Zellsäften einiger höherer Pflanzen

Ohne ZusammenfassungDie vorliegenden Versuehe wurden während meines kurzen Aufenthalts im Pflanzenphysiologisehen Institut der Universität Wien ausgeführt. Herrn Prof. Dr. Karl Höfler und Herrn Dr. H. Kinzel danke ich sehönstens auch an dieser Stelle für die Untersfützung während der Arbeit.     

Zur Kenntnis der Phlobaphenkörper in Früchten einigerMalus-Arten

Zusammenfassung Die von Molisch in der Epidermis und dem Hypoderm der Weinbeeren entdeckte Erscheinung, daß sich an der den Sonnenstrahlen ausgesetzten Seite dieser Früchte aus flüssigem Gerbstoff feste und gefärbte Phlobaphenballen bilden, wird auch in der Epidermis der Früchte vonMalus Toringo undM. floribunda beobachtet. Da — besonders beiMalus-Toringo- Früchten — unter der Einwirkung des Lichtes an der besonnten Fruchtseite reichlich auch Anthozyan erscheint, kommt es an dieser Seite zu einer mosaikartigen Verteilung der Zellen, von denen die einen feste, gelb bis gelbbraun gefärbte Phlobaphenkörper (verfestigte Vakuolen), die anderen flüssige, anthozyanführende Vakuolen enthalten. In manchen Fällen befinden sich beide Arten von Vakuolen in ein und derselben Zelle. Einige feste Vakuolen (Phlobaphenkörper) sind in einer intrazellulären Membran eingekapselt, deren Gerüstsubstanz Zellulosecharakter hat.In den flüssigen, ungefärbten Vakuolen, aus denen Phlobaphenkörper entstehen, wurde die Anwesenheit des Gerbstoffes sichergestellt. Es wird aber vermutet, daß den Gerbstoffen auch andere kolloide Substanzen beigemengt sind.Die Untersuchung wurde während meines dreimonatigen Aufenthaltes im Pflanzenphysiologischen Institut der Universität Graz durchgeführt. Dem Vorstand des Institutes, Herrn Professor Dr. F. Weber, sowie Herrn Professor Dr. O. Härtel und Frau Dr. L. Brat danke ich bestens für ihr liebenswürdiges Entgegenkommen.