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.     

The redox-switch domain of Hsp33 functions as dual stress sensor

The redox-regulated chaperone Hsp33 is specifically activated upon exposure of cells to peroxide stress at elevated temperatures. Here we show that Hsp33 harbors two interdependent stress-sensing regions located in the C-terminal redox-switch domain of Hsp33: a zinc center sensing peroxide stress conditions and an adjacent linker region responding to unfolding conditions. Neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33's full activation. Upon activation, Hsp33's redox-switch domain adopts a natively unfolded conformation, thereby exposing hydrophobic surfaces in its N-terminal substrate-binding domain. The specific activation of Hsp33 by the oxidative unfolding of its redox-switch domain makes this chaperone optimally suited to quickly respond to oxidative stress conditions that lead to protein unfolding.     

A logical model provides insights into T cell receptor signaling

Cellular decisions are determined by complex molecular interaction networks. Large-scale signaling networks are currently being reconstructed, but the kinetic parameters and quantitative data that would allow for dynamic modeling are still scarce. Therefore, computational studies based upon the structure of these networks are of great interest. Here, a methodology relying on a logical formalism is applied to the functional analysis of the complex signaling network governing the activation of T cells via the T cell receptor, the CD4/CD8 co-receptors, and the accessory signaling receptor CD28. Our large-scale Boolean model, which comprises 94 nodes and 123 interactions and is based upon well-established qualitative knowledge from primary T cells, reveals important structural features (e.g., feedback loops and network-wide dependencies) and recapitulates the global behavior of this network for an array of published data on T cell activation in wild-type and knock-out conditions. More importantly, the model predicted unexpected signaling events after antibody-mediated perturbation of CD28 and after genetic knockout of the kinase Fyn that were subsequently experimentally validated. Finally, we show that the logical model reveals key elements and potential failure modes in network functioning and provides candidates for missing links. In summary, our large-scale logical model for T cell activation proved to be a promising in silico tool, and it inspires immunologists to ask new questions. We think that it holds valuable potential in foreseeing the effects of drugs and network modifications.     

Protective Role of the Virus-Specific Immune Response for Development of Severe Neurologic Signs in Simian Immunodeficiency Virus-Infected Macaques

The pathogenesis of human immunodeficiency virus-associated motor and cognitive disorders is poorly understood. In this context both a protective and a harmful role of the immune system has been discussed. This question was addressed in the present study by correlating the occurrence of neurologic disease in simian immunodeficiency virus (SIV)-infected macaques with disease progression and the humoral and cellular intrathecal antiviral immune response. Overt neurologic signs consisting of ataxia and apathy were observed at a much higher frequency in rapid progressor animals (6 of 12) than in slow progressors (1 of 7). Whereas slow progressors mounted a strong antiviral antibody (Ab) response as evidenced by enzyme-linked immunosorbent and immunospot assays, neither virus-specific Ab titers nor Ab-secreting cells could be found in the cerebrospinal fluid (CSF) or brain parenchyma of rapid progressors. Similarly, increased infiltration of CD8⁺ T cells and cytotoxic T lymphocytes specific for viral antigens were detected only in the CSF of slow progressors. The finding that neurologic signs develop frequently in SIV-infected macaques in the absence of an antiviral immune response demonstrates that the immune system does not contribute to the development of motor disorders in these animals. Moreover, the lower incidence of neurologic symptoms in slow progressors with a strong intrathecal immune response suggests a protective role of the virus-specific immunity in immunodeficiency virus-induced central nervous system disease.     

Assignment and evaluation of ground beetle (Coleoptera: Carabidae) assemblages to sites on different scales in a grassland landscape

Carabid beetles and environmental parameters were investigated in 52 grassland sites with three replicate pitfall traps in each site and in the valley of the River Eider in Schleswig–Holstein (northern Germany) with 61 pitfall traps. Environmental parameters included water content of soil, sand content, organic matter content and pH. Ground beetle assemblages were derived by detrended correspondence analyses (DCA) and characterised by the specific environmental conditions as means for each assemblage. On the regional scale including all investigated sites of Schleswig–Holstein, five assemblages were differentiated. On the local scale including the investigated sites in the valley of the River Eider, three assemblages were found corresponding well with those found on the regional scale. Environmental conditions at the sites of the five assemblages were correlated with land use data, soil types, and water level stages provided by three maps of a geographic information system (GIS). The GIS maps were combined to develop smaller areas with land use, soil type and water level stage information. The characteristic environmental conditions were assigned to each area to derive the spatial distribution of the five ground beetle assemblages. Spatial prediction was correct for 65% of investigated sites. The potential area of each assemblage was estimated for the valley. The different grassland areas were evaluated as potential habitats for ground beetle species comparing total species richness with the regional species richness of each assemblage. The comparison shows that species richness in the evaluated assemblages is relatively low compared to the regional potential.     

Mechano-electric feedback and arrhythmias

The mechanical state of the heart feeds back to modify cardiac rate and rhythm. Mechanical stretch of myocardial tissue causes immediate and chronic responses that lead to the common end point of arrhythmia. This review provides a brief summary of the author's personal choice of contributions that she considers have fostered our understanding of the role of mechano-electric feedback in arrhythmogenesis.

Acute mechanical stretch reversibly depolarises the cell membrane and shortens the action potential duration. These electrophysiological changes are related to the activation of mechano-sensitive ion channels. Several different ion channels are involved in the sensing of stretch, among them K⁺-selective, Cl⁻-selective, non-selective, and ATP-sensitive K⁺ channels. Sodium and Ca²⁺ entering the cells via non-selective ion channels are thought to contribute to the genesis of stretch-induced arrhythmia. Mechano-sensitive channels have been cloned from non-vertebrate and vertebrate species.

Chronic stress on the heart activates gene expression in cardiomyocytes and non-myocytes. The signal transduction involves atrial natriuretic peptides and growth factors that initiate remodelling processes leading to hypertrophy which in turn may contribute to the electrical instability of the heart by increasing the responsiveness of mechano-sensitive channels. Selective block of these channels could provide some new form of treatment of mechanically induced arrhythmias, although at present there are no drugs available with sufficient selectivity. Detailed understanding of how mechanical strain on myocardial cells is translated into channel activation will allow to identify new targets for putative antiarrhythmic drugs.     

Understanding human cancer using Drosophila: Tid47, a cytosolic product of the DnaJ-like tumor suppressor gene l2Tid,is a novel molecular partner of patched related to skin cancer

Recessive mutations of the Drosophila gene lethal(2)-tumorous imaginal discs (l(2)tid) cause neoplastic growth of the anlagen of the adult organs, the imaginal discs. Here we report that the three proteins encoded by this evolutionarily conserved gene, Tid50, Tid47, and Tid40, identified as members of the DnaJ cochaperone family, are destined for different cellular compartments, build complexes with many proteins in a developmental stage-specific manner, and are likely to be involved in different cellular processes. We show that the cytosolic Tid47 molecule is a novel component of the Hedgehog (Hh)-Patched (Ptc) signaling regulating cell/tissue polarity and spatial patterning during development and is associated with human tumors such as basal cell carcinoma (BCC) and medulloblastoma. We provide functional evidence for its direct in vivo interaction with the Hh-bound Ptc receptor during signal transmission. Because loss of l(2)tid causes neoplastic transformation of Hh-responsive cells, we suggest that Tid47 may at least act as a guardian of the Hh signaling gradient by regulating Ptc homeostasis in the tissue. Finally, we show that the expression of htid-1, the human counterpart of l(2)tid, is altered in human BCCs. We demonstrate that in BCCs loss of htid expression correlates with loss of differentiation capacity of the neoplastic cells similar to that found in the Drosophila tumor model.     

Just the beginning: novel functions for angiotensin-converting enzymes

Cardiovascular disease is predicted to be the commonest cause of death worldwide by the year 2020. Diabetes, smoking and hypertension are the main risk factors. The renin-angiotensin system plays a key role in regulating blood pressure and fluid and electrolyte homeostasis in mammals. The discovery of specific drugs that block either the key enzyme of the renin-angiotensin system, angiotensin-converting enzyme (ACE), or the receptor for its main effector angiotensin II, was a major step forward in the treatment of hypertension and heart failure. In recent years, however, the renin-angiotensin system has been shown to be a far more complex system than initially thought. It has become clear that additional peptide mediators are involved. Furthermore, a new ACE, angiotensin-converting enzyme 2 (ACE2), has been discovered which appears to negatively regulate the renin-angiotensin system. In the heart, ACE2 deficiency results in severe impairment of cardiac contractility and upregulation of hypoxia-induced genes. We shall discuss the interplay of the various effector peptides generated by angiotensin-converting enzymes ACE and ACE2, highlighting the role of ACE2 as a negative regulator of the renin-angiotensin system.     

Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.