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Alzheimer's disease-related mutations in the presenilin-1 gene (PS1) are leading to an elevated production of neurotoxic beta-amyloid 1-42 and may additionally enhance oxidative stress. Here, we provide in vivo evidence indicating that brains of transgenic mice expressing different human Alzheimer-linked PS1 mutations exhibit a reduced activity of two antioxidant enzymes. For this purpose, mice transgenic for human PS1 and for single and multiple PS1 mutations were generated. Mice with multiple PS1 mutations showed a significantly decreased activity of the antioxidant enzymes Cu/Zn superoxide dismutase and glutathione reductase already at an age of 3-4 months. As expected, this effect was less pronounced for the mice with a single PS1 mutation. By contrast, animals bearing normal human PS1 showed significantly elevated enzyme activities relative to non-transgenic littermate controls.
Background Reliable taxonomic identification at the species level is the basis for many biological disciplines. In order to distinguish species, it is necessary that taxonomic characters allow for the separation of individuals into recognisable, homogeneous groups that differ from other such groups in a consistent way. We compared here the suitability and efficacy of traditionally used shell morphology and DNA-based methods to distinguish among species of the freshwater snail genus Radix (Basommatophora, Pulmonata). Results Morphometric analysis showed that shell shape was unsuitable to define homogeneous, recognisable entities, because the variation was continuous. On the other hand, the Molecularly defined Operational Taxonomic Units (MOTU), inferred from mitochondrial COI sequence variation, proved to be congruent with biological species, inferred from geographic distribution patterns, congruence with nuclear markers and crossing experiments. Moreover, it could be shown that the phenotypically plastic shell variation is mostly determined by the environmental conditions experienced. Conclusion Contrary to DNA-taxonomy, shell morphology was not suitable for delimiting and recognising species in Radix. As the situation encountered here seems to be widespread in invertebrates, we propose DNA-taxonomy as a reliable, comparable, and objective means for species identification in biological research.
Enhanced apoptosis and elevated levels of reactive oxygen species (ROS) play a major role in aging. In addition, several neurodegenerative diseases are associated with increased oxidative stress and apoptosis in neuronal tissue. Antioxidative treatment has neuro-protective effects. The aim of the present study was to evaluate changes of susceptibility to apoptotic cell death by oxidative stress in aging and its inhibition by the antioxidant Ginkgo biloba extract EGb761. We investigated basal and ROS-induced levels of apoptotic lymphocytes derived from the spleen in young (3 months) and old (24 months) mice. ROS were induced by 2-deoxy-D-ribose (dRib) that depletes the intracellular pool of reduced glutathione. Lymphocytes from aged mice accumulate apoptotic cells to a significantly higher extent under basal conditions compared to cells from young mice. Treatment with dRib enhanced this difference, implicating a higher sensitivity to ROS in aging. Apoptosis can be reduced in vitro by treatment with EGb761. In addition, mice were treated daily with 100mg/kg EGb761 per os over a period of two weeks. ROS-induced apoptosis was significantly reduced in the EGb761 group. Interestingly, this effect seemed to be more pronounced in old mice.
Apoptosis seems to be involved in immunosenescence associated with aging. Moreover, in lymphocytes (PBL) of patients with Alzheimer's disease, an increased susceptibility to the apoptotic pathway has been described possibly due to impaired protection of oxidative stress. Accordingly, it seemed to be of particular interest to investigate the contribution of normal aging to the susceptibility from human lymphocytes to programmed cell death. We could show that PBL from elderly individuals (>60 years) accumulate apoptosing cells to a significant higher extent in spontaneous and activation-induced cell death compared to younger controls (<35 years). Treatment with the oxidative stressor 2-deoxy-D-ribose or with agonistic-CD95-antibody pronounced this effect even more implicating a higher sensitivity to reactive oxygen species and a higher functional CD95 expression, respectively. In addition, expression of the activation markers HLA-DR and CD95 was significantly increased in CD3+-cells of aged subjects, while expression of CD25 did not seem to be affected by age. Expression of Bcl-2 was increased in aging and correlated with the number of apoptotic cells.
The relevance of physiological immune aging is of great interest with respect to determining disorders with pathologic immune function in aging individuals. In recent years, the relevance of changes in peripheral lymphocytes in age-associated neurologic diseases has become more evident. Due to the lack of immunological studies, covering more than one event after mitogenic activation, we envisaged a new concept in the present study, aiming to investigate several events, starting from T cell receptor (TCR) ligation up to T cell proliferation. In addition, we addressed the question whether changes are present in the subsets (CD4, CD8) with aging. Phosphorylation of tyrosine residues declines with increasing age in CD4+ cells. Fewer levels of CD69 positive cells after 4 h mitogenic activation, altered expression of cytokines (IL2, IFN-gamma and TNF-alpha; 22 h) and lower proliferation (72 h) were determined in aging. Moreover, it could be shown that CD8+ lymphocytes react more effectively to mitogenic stimulation with reference to CD69 expression and proliferation in both age groups (<35 and >60 years old). These data indicate that T cell activation, mediated by TCR engagement, is significantly impaired in aging and both subsets are affected. However, bypassing the TCR does not fully restore T cell function, indicating that there are more mechanisms involved than impaired signal transduction through TCR only. The results will be discussed in relation to their relevance in neurodegenerative and psychiatric disorders.
In vivo manipulation of interleukin-2 expression by a retroviral tetracycline (tet)-regulated system
(1999)
We have used the tetracycline (tet)-regulated system as described previously to evaluate the applicability of controlled gene expression in cancer gene therapy. As a model gene, we used the human interleukin-2 (IL-2) gene, which has been placed under the transcriptional control of the tetO/promoter. Human melanoma cells were transduced by two modified retroviral tet vectors containing the transactivator regulatory unit and the IL-2 gene driven by the tetO/promoter, respectively. In the absence of tet, IL-2 expression in the target cells was stable over several months. IL-2 production was in the range of 40 U/106 cells/24 hours. A fine tuning of IL-2 expression could be achieved by culturing the transduced cells with increasing doses of tet, whereby a concentration of 500 ng/mL tet in the culture medium abrogated IL-2 expression. Most importantly for clinical application, IL-2 expression by the transduced melanoma cells could also be regulated in vivo. When nu/nu mice were inoculated with the transduced tumor cells, they failed to develop tumors. Instead, the inhibition of IL-2 expression in the transduced tumor cells by oral administration of tet led to subcutaneous tumor growth; this growth rate was comparable with the growth rate of subcutaneously inoculated untransduced parental cells. The finding demonstrates the applicability of the tet-regulated system in cancer gene therapy.
Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Expression of PS1 mutations in cell culture systems and in primary neurons from transgenic mice increases their vulnerability to cell death. Interestingly, enhanced vulnerability to cell death has also been demonstrated for peripheral lymphocytes from AD patients. We now report that lymphocytes from PS1 mutant transgenic mice show a similar hypersensitivity to cell death as do peripheral cells from AD patients and several cell culture systems expressing PS1 mutations. The cell death-enhancing action of mutant PS1 was associated with increased production of reactive oxygen species and altered calcium regulation, but not with changes of mitochondrial cytochrome c. Our study further emphasizes the pathogenic role of mutant PS1 and may provide the fundamental basis for new efforts to close the gap between studies using neuronal cell lines transfected with mutant PS1, neurons from transgenic animals, and peripheral cells from AD patients. Copyright 2001 Academic Press.
In large models of neuronal cell death, there is a tight correlation between Cdk5 deregulation and cell-cycle dysfunction. However, pathways that link Cdk5 to the cell cycle during neuronal death are still unclear. We have investigated the molecular events that precede p25/Cdk5-triggered neuronal death using a neuronal cell line that allows inducible p25 expression. In this system, no sign of apoptosis was seen before 24 hours of p25 induction. Thus, at that time, cell-cycle-regulatory proteins were analysed by immunoblotting and some of them showed a significant deregulation. Interestingly, after time-course experiments, the earliest feature correlated with p25 expression was the phosphorylation of the retinoblastoma protein (Rb). Indeed, this phosphorylation was observed 6 hours after p25 induction and was abolished in the presence of a Cdk5 inhibitor, roscovitine, which does not inhibit the usual Rb cyclin-D kinases Cdk4 and Cdk6. Furthermore, analyses of levels and subcellular localization of Cdk-related cyclins did not reveal any change following Cdk5 activation, arguing for a direct effect of Cdk5 activity on Rb protein. This latter result was clearly demonstrated by in vitro kinase assays showing that the p25-Cdk5 complex in our cell system phosphorylates Rb directly without the need for any intermediary kinase activity. Hence, Rb might be an appropriate candidate that connects Cdk5 to cell-cycle deregulation during neuronal cell death.
The identification of specific genetic (presenilin-1 [PS1] and amyloid precursor protein [APP] mutations) and environmental factors responsible for Alzheimer's disease (AD) has revealed evidence for a shared pathway of neuronal death. Moreover, AD-specific cell defects may be observed in many other nonneuronal cells (e.g., lymphocytes). Thus, lymphocytes may serve as a cellular system in which to study risk factors of sporadic, as well as genetic AD in vivo. The aim of our present study was to clarify whether lymphocytes bearing genetic or sporadic risk factors of AD share an increased susceptibility to cell death. Additionally we examined whether a cell typespecific vulnerability pattern was present and how normal aging, the main risk factor of sporadic AD, contributes to changes in susceptibility to cell death. Here, we report that lymphocytes affected by sporadic or genetic APP and PS1 AD risk factors share an increased vulnerability to cell death and exhibit a similar cell type-specific pattern, given that enhanced vulnerability was most strongly developed in the CD4+ T-cell subtype. In this paradigm, sporadic risk factors revealed the highest impact on cell type-specific sensitivity of CD4+ T cells to apoptosis. In contrast, normal aging results in an increased susceptibility to apoptosis of both, CD4+ and CD8+ T cells.
Introduction: Reactive oxygen species (ROS) have been implicated in neurodegeneration and seem to be involved in the physiology and pathophysiology of several diseases, including normal aging and Alzheimer’s disease (AD). Enhanced ROS production in aging or AD is not restricted to the brain, but can also been seen in several peripheral tissues. The objective of the present study was to evaluate whether the mechanisms involved in the generation of oxidative stress in normal senescence and Alzheimer’s disease are identical or not. Methods: We analysed intracellular basal levels of ROS in lymphocytes from AD patients and healthy young and aged not-demented subjects as well as ROS levels following stimulation with d-ribose and staurosporine in all three groups. ROS levels were measured by flow cytometry using the intracellular fluorescence dye dihydrorhodamine123 (DHR123). Results: Our study shows that AD lymphocytes have increased basal levels of ROS, low susceptibility to ROS stimulation by 2-deoxy-D-ribose (dRib) and an increased response to staurosporine when compared with age-matched controls. Discussion: The data suggest that the defect(s) responsible for enhanced ROS production in AD may involve different or additional biological pathways than those involved in enhanced ROS generation during aging.
As major sources of reactive oxygen species (ROS), mitochondrial structures are exposed to high concentrations of ROS and may therefore be particularly susceptible to oxidative damage. Mitochondrial damage could play a pivotal role in the cell death decision. A decrease in mitochondrial energy charge and redox state, loss of transmembrane potential (depolarization), mitochondrial respiratory chain impairment, and release of substances such as calcium and cytochrome c all contribute to apoptosis. These mitochondrial abnormalities may constitute a part of the spectrum of chronic oxidative stress in Alzheimer's disease. Accumulation of amyloid beta (Abeta) in form of senile plaques is also thought to play a central role in the pathogenesis of Alzheimer's disease mediated by oxidative stress. In addition, increasing evidence shows that Abeta generates free radicals in vitro, which mediate the toxicity of this peptide. In our study, PC12 cells were used to examine the protective features of EGb 761(definition see editorial) on mitochondria stressed with hydrogen peroxide and antimycin, an inhibitor of complex III. In addition, we investigated the efficacy of EGb 761 in Abeta-induced MTT reduction in PC12 cells. Moreover, we examined the effects of EGb 761 on ROS levels and ROS-induced apoptosis in lymphocytes from aged mice after in vivo administration. Here, we will report that EGb 761 was able to protect mitochondria from the attack of hydrogen peroxide, antimycin and Abeta. Furthermore, EGb 761 reduced ROS levels and ROS-induced apoptosis in lymphocytes from aged mice treated orally with EGb 761 for 2 weeks. Our data further emphasize neuroprotective properties of EGb 761, such as protection against Abeta-toxicity, and antiapoptotic properties, which are probably due to its preventive effects on mitochondria.
FGF-2, a potent multifunctional and neurotrophic growth factor, is widely expressed in the brain and upregulated in cerebral ischemia. Previous studies have shown that intraventricularly or systemically administered FGF-2 reduces the size of cerebral infarcts. Whether endogenous FGF-2 is beneficial for the outcome of cerebral ischemia has not been investigated. We have used mice with a null mutation of the fgf2 gene to explore the relevance of endogenous FGF-2 in brain ischemia. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCAO). We found a 75% increase in infarct volume in fgf2 knock-out mice versus wild type littermates (P < 0.05). This difference in the extent of ischemic damage was observed after 24 h, and correlated with decreased viability in fgf2 mutant mice following MCA occlusion. Increased infarct volume in fgf2 null mice was associated with a loss of induction in hippocampal BDNF and trkB mRNA expression. These findings indicate that signaling through trkB may contribute to ameliorating brain damage following ischemia and that bdnf and trkB may be target genes of FGF-2. Together, our data provide the first evidence that endogenous FGF-2 is important in coping with ischemic brain damage suggesting fgf2 as one crucial target gene for new therapeutic strategies in brain ischemia.
Background The reciprocal (9;22) translocation fuses the bcr (breakpoint cluster region) gene on chromosome 22 to the abl (Abelson-leukemia-virus) gene on chromosome 9. Depending on the breakpoint on chromosome 22 (the Philadelphia chromosome – Ph+) the derivative 9+ encodes either the p40(ABL/BCR) fusion transcript, detectable in about 65% patients suffering from chronic myeloid leukemia, or the p96(ABL/BCR) fusion transcript, detectable in 100% of Ph+ acute lymphatic leukemia patients. The ABL/BCRs are N-terminally truncated BCR mutants. The fact that BCR contains Rho-GEF and Rac-GAP functions strongly suggest an important role in cytoskeleton modeling by regulating the activity of Rho-like GTPases, such as Rho, Rac and cdc42. We, therefore, compared the function of the ABL/BCR proteins with that of wild-type BCR. Methods We investigated the effects of BCR and ABL/BCRs i.) on the activation status of Rho, Rac and cdc42 in GTPase-activation assays; ii.) on the actin cytoskeleton by direct immunofluorescence; and iii) on cell motility by studying migration into a three-dimensional stroma spheroid model, adhesion on an endothelial cell layer under shear stress in a flow chamber model, and chemotaxis and endothelial transmigration in a transwell model with an SDF-1α gradient. Results Here we show that both ABL/BCRs lost fundamental functional features of BCR regarding the regulation of small Rho-like GTPases with negative consequences on cell motility, in particular on the capacity to adhere to endothelial cells. Conclusion Our data presented here describe for the first time an analysis of the biological function of the reciprocal t(9;22) ABL/BCR fusion proteins in comparison to their physiological counterpart BCR.
The interaction of T cells and antigen-presenting cells is central to adaptive immunity and involves the formation of immunological synapses in many cases. The surface molecules of the cells form a characteristic spatial pattern whose formation mechanisms and function are largely unknown. We perform computer simulations of recent experiments on geometrically repatterned immunological synapses and explain the emerging structure as well as the formation dynamics. Only the combination of in vitro experiments and computer simulations has the potential to pinpoint the kind of interactions involved. The presented simulations make clear predictions for the structure of the immunological synapse and elucidate the role of a self-organizing attraction between complexes of T cell receptor and peptide–MHC molecule, versus a centrally directed motion of these complexes.
In dieser Arbeit sollen zwei Themenkomplexe, jeder für sich außerordentlich interessant und vielschichtig, betrachtet und verknüpft werden. Das direkte Einbinden von lebenden Tieren in die Lehre des Lebendigen ist nicht nur sinnvoll, sondern wichtig und wird auch von den Rahmenplänen Biologie empfohlen. Mit dem Einsatz von Tieren im Unterricht wird sich an verschiedenen Stellen beschäftigt (z.B. OGILVIE & STINSON 1995). Der Klasse Reptilia kommt dabei aber vergleichsweise wenig Beachtung zu. Voraussetzung für die Haltung von Tieren in der Schule, die in der Öffentlichkeit steht und eine beachtliche Vorbildsfunktion inne hat, ist unter anderem, dass eine „artgerechte“ Unterbringung und Pflege möglich ist. Was unter der Bezeichnung einer „artgerechten Tierhaltung“ verstanden wird, ist alles andere als eindeutig. Deshalb soll die Thematik mit Bezug auf die Reptilienhaltung in der Schule beleuchtet werden.
The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene’s influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT–PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2{Delta}, rlf2{Delta} and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2{Delta} pso4-1 and rlf2{Delta} pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.
Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-ß-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-ß-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable ‘base-paired’ geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair.
The radiation-sensitive mutant pso4-1 of Saccharomyces cerevisiae shows a pleiotropic phenotype, including sensitivity to DNA cross-linking agents, nearly blocked sporulation and reduced mutability. We have cloned the putative yeast DNA repair gene PSO4 from a genomic library by complementation of the blocked UV-induced mutagenesis and of sporulation in diploids homozygous for pso4-1. Sequence analysis revealed that gene PSO4 consists of 1512 bp located upstream of UBI4 on chromosome XII and encodes a putative protein of 56.7 kDa. PSO4 is allelic to PRP19, a gene encoding a spliceosome-associated protein, but shares no significant homology with other yeast genes. Gene disruption with a destroyed reading frame of our PSO4 clone resulted in death of haploid cells, confirming the finding that PSO4/PRP19 is an essential gene. Thus, PSO4 is the third essential DNA repair gene found in the yeast S.cerevisiae.
Das Thema der vorliegenden Arbeit war die molekulargenetische Charakterisierung der Funktion der Glukosesensoren Snf3 und Rgt2 in der Hefe S. cerevisiae. Snf3 und Rgt2 gehören zur Familie der Hexosetransporter. Sie unterscheiden sich von ihnen jedoch in ihrer Funktion als Glukosesensoren wie auch durch ihre ungewöhnlich langen Cterminalen Domänen. Snf3 und Rgt2 sind integrale Membranproteine, die als Reaktion auf extrazelluläre Glukose Signale auslösen, die zur Expression bestimmter Hexosetransporter führt. Einige Komponenten, die an der Signaltranduktion beteiligt sind, wurden bereits identifiziert. Jedoch ist der genaue Mechanismus, der zur Expression der Hexostransporter führt, noch nicht vollständig aufgeklärt. Im ersten Teil dieser Arbeit wurden die Proteine Snf3, Rgt2, Mth1, Std1 und Rgt1 auf direkte Interaktionen untereinander getestet, um Einblicke in den molekularen Mechanismus der Signaltransduktion zu erhalten. Desweiteren sollte festgestellt werden, ob die Protein-Wechselwirkungen von der C-Quelle abhängig sind. Es konnte gezeigt werden, dass zwischen den Membranproteinen Rgt2 bzw. Snf3 und den löslichen Proteinen Mth1 bzw. Std1 Interaktionen in Abhängigkeit von Glukose stattfanden. Diese Ergebnisse unterstützen das von Moriya und Johnston aufgestellte, gegenwärtige Modell für eine glukoseinduzierte HXT Genexpression. Im zweiten Teil dieser Arbeit wurde geprüft, ob sich aus dem Glukosesensor Snf3 durch eine Aminosäuresubstitution ein bifunktionaler Sensor für Glukose und Galaktose erzeugen läßt. Dazu wurden die für den Galaktosetransport verantwortlichen Aminosäuren in den homologen Positionen von Snf3 ausgetauscht. Die Bestimmungen der Regulation des Snf3-kontrollierten HXT7 Promotors ergaben, dass das mutierte Snf3 Protein, wie das Wildtyp-Snf3 Protein, eine normale Glukosesensorfunktion ausübt aber keine Galaktosesensorfunktion vorzeigt.
Background: The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. Results: N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. Conclusion: The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base.