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.     

Myeloid-derived growth factor regulates neutrophil motility in interstitial tissue damage

Neutrophil recruitment to tissue damage is essential for host defense but can also impede tissue repair. The cues that differentially regulate neutrophil responses to tissue damage and infection remain unclear. Here, we report that the paracrine factor myeloid-derived growth factor (MYDGF) is induced by tissue damage and regulates neutrophil motility to damaged, but not infected, tissues in zebrafish larvae. Depletion of MYDGF impairs wound healing, and this phenotype is rescued by depleting neutrophils. Live imaging and photoconversion reveal impaired neutrophil reverse migration and inflammation resolution in mydgf mutants. We found that persistent neutrophil inflammation in tissues of mydgf mutants was dependent on the HIF-1α pathway. Taken together, our data suggest that MYDGF is a damage signal that regulates neutrophil interstitial motility and inflammation through a HIF-1α pathway in response to tissue damage.     

O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: studies addressing safety issues

As haematopoietic stem cell gene therapy utilizing O(6)-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O(6) alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level.     

Purine nucleosides support the neurite outgrowth of primary rat cerebellar granule cells after hypoxia

Mammalian neurons require a constant supply of oxygen to maintain adequate cellular functions and survival. Following sustained hypoxia during ischemic events in brain, the energy status of neurons and glia is compromised, which may subsequently lead to cell death by apoptosis and necrosis. Concomitant with energy depletion is the formation of the purine nucleoside adenosine, a powerful endogenous neuroprotectant. In this paper the effect of chemical hypoxia on cell survival and neurite outgrowth of primary cerebellar granule cells was investigated. Rotenone, a mitochondrial complex I inhibitor, induced a 30.4 +/- 3.6% loss of viable cells and a 35.0 +/- 4.4% loss of neurite formation of cerebellar granule cells, which was partially restored by the addition of purine nucleosides adenosine, inosine and guanosine. Inosine had the most striking effect of 37.7 +/- 2.9% rescue of viability and 71.2 +/- 18.4% rescue of neurite outgrowth. Data confirm the suggested role of purine nucleosides for the neuronal regeneration of primary brain cells following hypoxic insult.     

A preliminary phylogeny of the 'didymocarpoid Gesneriaceae' based on three molecular data sets: Incongruence with available tribal classifications

The 'didymocarpoid Gesneriaceae' (traditional subfam. Cyrtandroideae excluding Epithemateae) are the largest group of Old World Gesneriaceae, comprising 85 genera and 1800 species. We attempt to resolve their hitherto poorly understood generic relationships using three molecular markers on 145 species, of which 128 belong to didymocarpoid Gesneriaceae. Our analyses demonstrate that consistent topological relationships can be retrieved from data sets with missing data using subsamples and different combinations of gene sequences. We show that all available classifications in Old World Gesneriaceae are artificial and do not reflect natural relationships. At the base of the didymocarpoids are grades of clades comprising isolated genera and small groups from Asia and Europe. These are followed by a clade comprising the African and Madagascan genera. The remaining clades represent the advanced Asiatic and Malesian genera. They include a major group with mostly twisted capsules. The much larger group of remaining genera comprises exclusively genera with straight capsules and the huge genus Cyrtandra with indehiscent fruits. Several genera such as Briggsia, Henckelia, and Chirita are not monophyletic; Chirita is even distributed throughout five clades. This degree of incongruence between molecular phylogenies, traditional classifications, and generic delimitations indicates the problems with classifications based on, sometimes a single, morphological characters.     

Evaluating the effectiveness of retention forestry to enhance biodiversity in production forests of Central Europe using an interdisciplinary,multi‐scale approach

Retention forestry, which retains a portion of the original stand at the time of harvesting to maintain continuity of structural and compositional diversity, has been originally developed to mitigate the impacts of clear‐cutting. Retention of habitat trees and deadwood has since become common practice also in continuous‐cover forests of Central Europe. While the use of retention in these forests is plausible, the evidence base for its application is lacking, trade‐offs have not been quantified, it is not clear what support it receives from forest owners and other stakeholders and how it is best integrated into forest management practices. The Research Training Group ConFoBi (Conservation of Forest Biodiversity in Multiple‐use Landscapes of Central Europe) focusses on the effectiveness of retention forestry, combining ecological studies on forest biodiversity with social and economic studies of biodiversity conservation across multiple spatial scales. The aim of ConFoBi is to assess whether and how structural retention measures are appropriate for the conservation of forest biodiversity in uneven‐aged and selectively harvested continuous‐cover forests of temperate Europe. The study design is based on a pool of 135 plots (1 ha) distributed along gradients of forest connectivity and structure. The main objectives are (a) to investigate the effects of structural elements and landscape context on multiple taxa, including different trophic and functional groups, to evaluate the effectiveness of retention practices for biodiversity conservation; (b) to analyze how forest biodiversity conservation is perceived and practiced, and what costs and benefits it creates; and (c) to identify how biodiversity conservation can be effectively integrated in multi‐functional forest management. ConFoBi will quantify retention levels required across the landscape, as well as the socio‐economic prerequisites for their implementation by forest owners and managers. ConFoBi's research results will provide an evidence base for integrating biodiversity conservation into forest management in temperate forests.     

The crossroads of neuroinflammation in infectious diseases: endothelial cells and astrocytes

Homeostasis implies constant operational defence mechanisms, against both external and internal threats. Infectious agents are prominent among such threats. During infection, the host elicits the release of a vast array of molecules and numerous cell-cell interactions are triggered. These pleiomorphic mediators and cellular effects are of prime importance in the defence of the host, both in the systemic circulation and at sites of tissue injury, for example, the blood-brain barrier (BBB). Here, we focus on the interactions between the endothelium, astrocytes, and the molecules they release. Our review addresses these interactions during infectious neurological diseases of various origins, especially cerebral malaria (CM). Two novel elements of the interplay between endothelium and astrocytes, microparticles and the kynurenine pathway, will also be discussed.