Showing papers by "Heidelberg University published in 1999"

Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease.

Abstract: Genetic evidence strongly supports the view that Aβ amyloid production is central to the cause of Alzheimer's disease. The kinetics, compartmentation, and form of Aβ and its temporal relation to the neurodegenerative process remain uncertain. The levels of soluble and insoluble Aβ were determined by using western blot techniques, and the findings were assessed in relation to indices of severity of disease. The mean level of soluble Aβ is increased threefold in Alzheimer's disease and correlates highly with markers of disease severity. In contrast, the level of insoluble Aβ (also a measure of total amyloid load) is found only to discriminate Alzheimer's disease from controls, and does not correlate with disease severity or numbers of amyloid plaques. These findings support the concept of several interacting pools of Aβ, that is, a large relatively static insoluble pool that is derived from a constantly turning over smaller soluble pool. The latter may exist in both intracellular and extracellular compartments, and contain the basic forms of Aβ that cause neurodegeneration. Reducing the levels of these soluble Aβ species by threefold to levels found in normal controls might prove to be a goal of future therapeutic intervention.

Nephropathy in Patients with Type 2 Diabetes Mellitus

Abstract: In the not-so-distant past, type 2 diabetes mellitus was thought to be a relatively benign condition, at least in the elderly, with relatively little effect on life expectancy or renal function.1 It has now become obvious that type 2 diabetes must be taken every bit as seriously as type 1 diabetes, in part because of its renal complications.2 However, some recent and encouraging evidence indicates that diabetic nephropathy and deterioration of renal function are to a certain extent preventable. Epidemiology According to the reports of the U.S. Renal Data System,3,4 in the past two decades there has been a . . .

Hemorrhagic Transformation Within 36 Hours of a Cerebral Infarct Relationships With Early Clinical Deterioration and 3-Month Outcome in the European Cooperative Acute Stroke Study I (ECASS I) Cohort

Abstract: Background and Purpose—The clinical correlates of the varying degrees of early hemorrhagic transformation of a cerebral infarct are unclear. We investigated the cohort of a randomized trial of thrombolysis to assess the early and late clinical course associated with different subtypes of hemorrhagic infarction (HI) and parenchymal hematoma (PH) detected within the first 36 hours of an ischemic stroke. Methods—We exploited the database of the European Cooperative Acute Stroke Study I (ECASS I), a randomized, placebo-controlled, phase III trial of intravenous recombinant tissue plasminogen activator in acute ischemic stroke. Findings on 24- to 36- hour CT were classified into 5 categories: no hemorrhagic transformation, HI types 1 and 2, and PH types 1 and 2. We assessed the risk of concomitant neurological deterioration and of 3-month death and disability associated with subtypes of hemorrhagic transformation, as opposed to no bleeding. Risks were adjusted for age and extent of ischemic damage on baseline ...

Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating

Abstract: High spatial frequencies in the illuminating light of microscopes lead to a shift of the object spatial frequencies detectable through the objective lens. If a suitable procedure is found for evaluation of the measured data, a microscopic image with a higher resolution than under flat illumination can be obtained. A simple method for generation of a laterally modulated illumination pattern is discussed here. A specially constructed diffraction grating was inserted in the illumination beam path at the conjugate object plane (position of the adjustable aperture) and projected through the objective into the object. Microscopic beads were imaged with this method and evaluated with an algorithm based on the structure of the Fourier space. The results indicate an improvement of resolution.

NF-kappaB is activated and promotes cell death in focal cerebral ischemia.

Abstract: The transcription factor NF-kappaB is a regulator of cell death or survival. To investigate the role of NF-kappaB in neuronal cell death, we studied its activation in a rodent model of stroke. In the ischemic hemisphere, NF-kappaB was activated, as determined by increased expression of an NF-kappaB-driven reporter transgene, nuclear translocation of NF-kappaB in neurons and enhanced DNA binding of NF-kappaB subunits RelA and p50. In p50 knockout mice, ischemic damage was significantly reduced. This indicates a cell death-promoting role of NF-kappaB in focal ischemia. NF-kappaB may provide a new pharmacological target in neurologic disease.

Comparison of Methods Based on Different Molecular Epidemiological Markers for Typing of Mycobacterium tuberculosis Complex Strains: Interlaboratory Study of Discriminatory Power and Reproducibility

Abstract: In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity Therefore, 90 M tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays In all methods, one or more repetitive DNA elements were targeted The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method To examine intralaboratory reproducibility, blinded duplicate samples were included The specificities of the various methods were tested by inclusion of 10 non-M tuberculosis complex strains All five RFLP typing methods were highly reproducible The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns) We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice A strong association was found between the results of different genetic markers, indicating a clonal population structure of M tuberculosis strains Several separate genotype families within the M tuberculosis complex could be recognized on the basis of the genetic markers used

The Dysregulated Podocyte Phenotype A Novel Concept in the Pathogenesis of Collapsing Idiopathic Focal Segmental Glomerulosclerosis and HIV-Associated Nephropathy

Abstract: Podocytes are highly differentiated, postmitotic cells, whose function is largely based on their complex cytoarchitecture. The differentiation of podocytes coincides with progressive expression of maturity markers, including WT-1, CALLA, C3b receptor, GLEPP-1, podocalyxin, and synaptopodin. In collapsing forms of focal segmental glomerulosclerosis (FSGS), including idiopathic FSGS and HIV-associated nephropathy, podocytes undergo characteristic, irreversible ultrastructural changes. This study analyzes the expression pattern of the above differentiation markers and of the proliferation marker Ki-67 in collapsing idiopathic FSGS and HIV-associated nephropathy compared with minimal change disease, membranous glomerulopathy, as well as normal adult and fetal human kidney. In minimal change disease and membranous glomerulopathy, all mature podocyte markers were retained at normal levels despite severe proteinuria and foot process fusion; no cell proliferation was observed. In contrast, in collapsing idiopathic FSGS and HIV-associated nephropathy, there was disappearance of all markers from all collapsed glomeruli and of synaptopodin from 16% of noncollapsed glomeruli. This phenotypic dysregulation of podocytes was associated with cell proliferation in both diseases. It is concluded that the loss of specific podocyte markers defines a novel dysregulated podocyte phenotype and suggests a common pathomechanism in collapsing FSGS, whether idiopathic or HIV-associated.

Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid

Abstract: Endophilin I is a presynaptic protein of unknown function that binds to dynamin, a GTPase that is implicated in endocytosis and recycling of synaptic vesicles. Here we show that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane. Endophilin I exhibits lysophosphatidic acid acyl transferase (LPAAT) activity, and endophilin-I-mediated SLMV formation requires the transfer of the unsaturated fatty acid arachidonate to lysophosphatidic acid, converting it to phosphatidic acid. A deletion mutant lacking the SH3 domain through which endophilin I interacts with dynamin still exhibits LPAAT activity but no longer mediates SLMV formation. These results indicate that endophilin I may induce negative membrane curvature by converting an inverted-cone-shaped lipid to a cone-shaped lipid in the cytoplasmic leaflet of the bilayer. We propose that, through this action, endophilin I works with dynamin to mediate synaptic vesicle invagination from the plasma membrane and fission.

Angiotensin Induces Inflammatory Activation of Human Vascular Smooth Muscle Cells

Abstract: Multiple data suggest that the renin-angiotensin system contributes to the pathogenesis of atherosclerosis. The atherogenic effect of the renin-angiotensin system can only in part be explained by the influence of its effector angiotensin II on blood pressure, smooth muscle cell (SMC) growth, or antifibrinolytic activity. Because chronic inflammation of the vessel wall is a hallmark of atherosclerosis, we hypothesized that angiotensin II may elicit inflammatory signals in vascular SMCs. Human vascular SMCs were stimulated with angiotensin. Inflammatory activation was assessed by determination of interleukin-6 (IL-6) release into the culture medium, detection of IL-6 mRNA by RT-PCR, and demonstration of activation of nuclear factor-kappaB in electrophoretic mobility shift assays. Angiotensin II concentration-dependently (1 nmol/L to 1 micromol/L) stimulated IL-6 production by SMCs via activation of the angiotensin II type 1 receptor (demonstrated by the inhibitory action of the receptor antagonist losartan). Angiotensin I increased IL-6 production by SMCs, too. This effect was inhibited by captopril and ramiprilat, suggesting conversion of angiotensin I to angiotensin II by angiotensin-converting enzyme in SMCs. Steady-state mRNA for IL-6 was augmented after stimulation with angiotensin II, suggesting regulation of angiotensin-induced IL-6 release at the pretranslational level. Moreover, the proinflammatory transcription factor nuclear factor-kappaB, which is necessary for transcription of most cytokine genes, was also activated by angiotensin II. Pyrrolidine dithiocarbamate suppressed angiotensin II-induced IL-6 release, a finding compatible with involvement of reactive oxygen species as second messengers in cytokine production mediated by angiotensin. The data demonstrate the ability of angiotensin to elicit an inflammatory response in human vascular SMCs by stimulation of cytokine production and activation of nuclear factor-kappaB. Inflammatory activation of the vessel wall by a dysregulated renin-angiotensin system may contribute to the pathogenesis of atherosclerosis.

Electrospray Ionization Tandem Mass Spectrometry (Esi-Ms/Ms) Analysis of the Lipid Molecular Species Composition of Yeast Subcellular Membranes Reveals Acyl Chain-Based Sorting/Remodeling of Distinct Molecular Species En Route to the Plasma Membrane

Abstract: Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of ∼± 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Δ mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.

CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons.

Abstract: Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

Detection of high-risk cervical intraepithelial neoplasia and cervical cancer by amplification of transcripts derived from integrated papillomavirus oncogenes.

Abstract: Cervical cancer emerges from cervical intraepithelial neoplasia (CIN) induced by high-risk HPV (HR-HPV) infections. However, the vast majority of CIN lesions regresses spontaneously, and only a few lesions persist or progress to invasive carcinoma. On the basis of morphological criteria, it is not possible to differentiate high-grade lesions that will regress or persist from those that inevitably will progress to invasive cancers. In most cervical carcinomas, human papillomavirus (HPV) genomes are integrated into host cell chromosomes and transcribed into mRNAs encompassing viral and cellular sequences. In contrast, in early preneoplastic lesions, HPV genomes persist as episomes, and derived transcripts contain exclusively viral sequences. Thus, detection of HPV transcripts derived from integrated HPV genomes may specifically indicate both CIN lesions at high risk for progression as well as invasive cervical cancers. Here, we established a protocol for the amplification of papillomavirus oncogene transcripts (APOT) from cervical specimens that allows us to distinguish episome- from integrate-derived HPV mRNAs. Cervical swab and biopsy samples from 549 patients attending outpatient clinics for cervical dysplasia were screened for the presence of HPV DNA, and 155 samples that were positive for either HPV type 16 (n = 143) or 18 (n = 12) were subjected to the APOT assay. In samples derived from normal cervical epithelia (n = 19) or low-grade cervical lesions (CIN I, n = 10), no integrate-derived HPV transcripts were found. In contrast, in 1 (5%) of 22 samples derived from CIN II lesions, in 10 (16%) of 64 samples from patients with CIN III lesions, and in 35 (88%) of 40 samples from patients with cervical cancer, integrate-derived HPV transcripts were detected. Thus, detection of integrate-derived HPV transcripts in cervical swabs or biopsy specimens by the APOT assay points to advanced dysplasia or invasive cervical cancer.

Pathophysiologic basis of contrast enhancement in breast tumors.

Abstract: While the diagnostic benefits of gadolinium (Gd)-chelate contrast agents are firmly established in magnetic resonance imaging (MRI) of tumors, the pathophysiologic basis of the enhancement observed and its histopathologic correlate remained vague. Tumor angiogenesis is fundamental for growth and metastasis and also of interest in new therapeutic concepts. By correlative analysis of a) histology; b) vascular density (CD31); and c) vascular permeability (vascular permeability factor/vascular endothelial growth factor [VPF/VEGF]), we found a) significantly (P F 0.001) faster exchange rates in malignant compared with benign breast lesions; b) distinct differences in enhancement characteristics between the histologic types (invasive ductal carcinoma, invasive lobular carcinoma, and ductal carcinoma in situ); and c) dependence of enhancement kinetics on the VPF/VEGF expression. The pathophysiologic basis for the differences in contrast enhancement patterns of tumors detectable by MRI is mainly due to vascular permeability, which leads to more characteristic differences than vascular density. MRI is able to subclassify malignant breast tumors due to their different angiogenetic properties. J. Magn. Reson. Imaging 1999; 10:260‐266. r 1999 Wiley-Liss, Inc.

The role of oxidative stress and NF-κB activation in late diabetic complications

Abstract: A common endpoint of hyperglycemia dependent cellular changes is the generation of reactive oxygen intermediates (ROIs) and the presence of elevated oxidative stress. Therefore, oxidative stress is supposed to play an important role in the development of late diabetic complications. Formation of advanced glycation end products (AGE's) due to elevated nonenzymatic glycation of proteins, lipids and nucleic acids is accompanied by oxidative, radical-generating reactions and thus represents a major source for oxygen free radicals under hyperglycemic conditions. Once formed, AGE's can influence cellular function by binding to several binding sites including the receptor for AGE's, RAGE. Binding of AGE's (and other ligands) to RAGE results in generation of intracellular oxidative stress and subsequent activation of the redox-sensitive transcription factor NF-kappaB in vitro and in vivo. Consistently, activation of NF-kappaB in diabetic patients correlates with the quality of glycemic control and can be reduced by treatment with the antioxidant alpha-lipoic acid. The development of techniques allowing for a tissue culture independent measurement of NF-kappaB activation in patients with diabetes mellitus gives insights into the molecular mechanisms linking diabetes mellitus and hyperglycemia with formation of advanced glycated endproducts and generation of oxidative stress finally resulting in oxidative stress mediated cellular activation.

Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 CONCERTED ACTION: investigation of minimal residual disease in acute leukemia.

Abstract: It is now widely accepted that the detection of minimal residual disease (MRD) has prognostic value in acute leukemia. However clinical MRD studies need standardized techniques. Therefore, several European laboratories have aligned their goals and performed comparative studies to achieve optimization and standardization of MRD techniques. This was achieved via the BIOMED-1 Concerted Action “Investigation of minimal residual disease in acute leukemia: International standardization and clinical evaluation.” This report describes the development of PCR primers and protocols for the detection of MRD in acute lymphoblastic leukemia (ALL) using clone-specific junctional regions of immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets. A total of 54 primers was developed (1) to amplify rearrangements of the TCRD, TCRG, and IGK (Kde) genes as well as TAL1 deletions; (2) to sequence the junctional regions and breakpoint fusion regions; and (3) to perform MRD detection in bone marrow or peripheral blood samples during follow-up of ALL patients. Protocols were established to identify PCR targets at diagnosis by performing 25 PCR reactions per patient using appropriate positive and negative controls. Standardized protocols were developed for MRD monitoring via single amplification of the PCR target followed by dot blot hybridization with the corresponding patient-specific junctional region probe. In addition, alternative approaches were designed for cases where the target sensitivity of at least 10−4 was not obtained. The standardization described here of MRD-PCR techniques is essential for the process of translating MRD research into clinical practice.

Acclimation of Arabidopsis leaves developing at low temperatures. Increasing cytoplasmic volume accompanies increased activities of enzymes in the Calvin cycle and in the sucrose-biosynthesis pathway.

Abstract: Photosynthetic and metabolic acclimation to low growth temperatures were studied in Arabidopsis (Heynh.). Plants were grown at 23 degrees C and then shifted to 5 degrees C. We compared the leaves shifted to 5 degrees C for 10 d and the new leaves developed at 5 degrees C with the control leaves on plants that had been left at 23 degrees C. Leaf development at 5 degrees C resulted in the recovery of photosynthesis to rates comparable with those achieved by control leaves at 23 degrees C. There was a shift in the partitioning of carbon from starch and toward sucrose (Suc) in leaves that developed at 5 degrees C. The recovery of photosynthetic capacity and the redirection of carbon to Suc in these leaves were associated with coordinated increases in the activity of several Calvin-cycle enzymes, even larger increases in the activity of key enzymes for Suc biosynthesis, and an increase in the phosphate available for metabolism. Development of leaves at 5 degrees C also led to an increase in cytoplasmic volume and a decrease in vacuolar volume, which may provide an important mechanism for increasing the enzymes and metabolites in cold-acclimated leaves. Understanding the mechanisms underlying such structural changes during leaf development in the cold could result in novel approaches to increasing plant yield.