The renin-angiotensin system in diabetic nephropathy.
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Angiotensin-Converting Enzyme Inhibitors↗
Biomedical subjects
G Wolf
Publications and source records attributed to G Wolf.
[Osteomalacia in vitamin D deficiency in a 32-year-old immigrant: case report of a rare disease].
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Adult↗
Thomas Mann's the Transposed Heads: an accidental allogenic transplantation with unfavorable outcome.
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Brain Tissue Transplantation↗
[The asphyxia-cardiac arrest rat model for developing mechanism of effect oriented therapeutic concepts after cardiopulmonary resuscitation].
In order to expand and combine clinical and basic science research opportunities on cardiac arrest, the Asphyxial Cardiac Arrest Rat Model, originally developed at the International Resuscitation Research Center in Pittsburgh, was introduced and adapted at the University of Magdeburg Medical Center. For better utilization of established morphological and biochemical evaluation techniques, the former Sprague-Dawley rat model was adapted for Wistar rats. Life-support procedures, especially controlled ventilation, had to be adjusted. Wistar rats seem to be more sensitive to asphyxia, resulting in significantly faster-developing cardiac arrest (time from ceasing ventilation to pulselessness: Wistar 2:43 min versus 3:28 min in Sprague-Dawley) and a tendentiously longer resuscitation time (37 versus 23 s). Furthermore, a trend towards a more pronounced secondary blood pressure depression was observed especially during the later half of the first hour after resuscitation. Post-mortem brain evaluations using conventional Nissl and haematoxylin-eosin staining techniques showed--most distinctly in the hippocampal CA1 region--delayed neuronal damage with a peak at 72 hours after resuscitation and with only a few neurons remaining after seven days of survival.
Animals↗
Differential expression of alternatively spliced isoforms of neuronal nitric oxide synthase (nNOS) and N-methyl-D-aspartate receptors (NMDAR) in knockout mice deficient in nNOS alpha (nNOS alpha(Delta/Delta) mice).
Recent data suggest that the neuronal isoform of nitric oxide synthase (nNOS) and glutamate receptors of the N-methyl-D-aspartate (NMDA) type are physically coupled and, hence, functionally interrelated. Several alternatively spliced isoforms of the N-methyl-D-aspartate receptor 1 (NMDAR1) subunit and the neuronal nitric oxide synthase (nNOS) are known, and recent studies have shown that a spliced C-terminal may be responsible for the coupling of NMDAR's to nNOS via its PDZ domain and the postsynaptic density protein PSD95. However, little is known about whether and to what extent changes in nNOS expression influence NMDA receptor density or function. We have therefore compared the localization of nNOS alpha, beta and gamma with that of two relevant NMDAR1 splice variants in wild-type mice versus knockout mice deficient in nNOS alpha, generated by homologous recombination with a targeted deletion of exon 2, containing one PDZ domain (nNOS alpha(Delta/Delta) mice). Whereas nNOS alpha was completely absent in nNOS alpha(Delta/Delta) mice, nNOS beta and gamma were expressed in both wild-type and knockout animals. nNOS gamma mRNA, though, was hardly detectable, if at all, mainly within the olfactory bulb, the cerebellum and mesencephalic nuclei of knockout animals. The expression of the NMDAR1-1 splice variant (without any short carboxy-terminal amino acid motif, recognized by PDZ domains) was remarkably decreased in striatal, cortical, hippocampal and cerebellar tissue in nNOS alpha(Delta/Delta) animals, but no changes in NMDAR1-4 (with an alternatively spliced C-terminal and thus with a PDZ binding motif) mRNA and protein levels were observed. While NMDAR1-4 may be related to receptor targeting and clustering to PSD95 and to nNOS, our data suggest that differences in nNOS expression obviously do not directly influence gene expression of this particular NMDAR splice variant. Otherwise, the observed diminution of NMDAR1-1 splice variant mRNA and protein levels may, at least partially, explain the decreased vulnerability of nNOS alpha(Delta/Delta) mice to glutamate-mediated neurotoxicity.
Alternative Splicing↗
"Show your Wound" medicine and the work of Joseph Beuys.
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Germany↗
Neuroma: a donor-age independent source of human Schwann cells for tissue engineered nerve grafts.
Schwann cells are used in combination with biological matrices as tissue engineered nerve grafts in animal models offering a new therapeutic approach for treatment of lesions of the peripheral nervous system. A high yield of human Schwann cells from adult donors is only achieved by pharmacological stimulation, which should, however, be avoided in clinical therapy. Here, we establish cultures of activated human Schwann cells which were isolated from peripheral nerve neuroma which developed after a median nerve lesion. To allow nerve reconstruction neuroma have to be resected. Such neuroma tissue is virtually predegenerated and shows activation of Schwann cells, implying good adherence and high mitotic activity. This allows, irrespective of donor age, growing within a short time period and without any pharmacological treatment.
Adult↗
A new inactivated BVDV genotype I and II vaccine. An immunisation and challenge study with BVDV genotype I.
An inactivated vaccine containing BVDV I and II strains (PT810; BVDV I, and 890; BVDV II) and using different adjuvants and antigen dosages was tested in a cattle challenge model. Groups of six healthy, seronegative cattle were vaccinated twice with a low dose (10(6.6) TCID(50) PT810 and 10(7.2) TCID(50) 890) vaccine with the adjuvant Bay R1005 or a high dose (10(7.8) TCID(50) PT810 and 10(8. 2) TCID(50) 890) vaccine with two different adjuvants (Bay R1005 or Polygen). Thirty-eight days after the second vaccination, immunised animals (n=18) and non-vaccinated control animals (n=3) were challenged intranasally with 10(6) TCID(50) BVDV strain PT810. For a period of 16 days, virus was isolated from blood leukocytes and nasal swabs, and neutralising antibody titres were determined.The induction of antibodies following immunisation was strongly dependent on the antigen dosage in the vaccine. The high dose formulation induced high serum neutralising antibody titres against both genotypes of up to 32000 after the second immunisation. Animals with neutralising antibody titres >512 (n=14) did not show any marked leukopenia after challenge and only very little or no virus could be isolated from blood leukocytes and/or nasal swabs when compared to control cattle. Furthermore, some of these animals did not show any boost of neutralising or even NS3-specific antibodies, which renders viral replication unlikely and thus would prevent infection of the fetus. Both adjuvants (Bay R1005 or Polygen) were similarly efficient and induced nearly identical antibody responses. In contrast, four of the six low dosage vaccinates had a marked leukopenia and viraemia as well as detectable nasal virus shedding for several days. We conclude that the selected strains and the system of vaccine preparation with high BVDV antigen dosages and highly efficient new adjuvants provide an effective means of protection against BVDV I infections. Investigations to demonstrate the protection against BVDV II infections, the duration of immunity and the ability of fetal protection by using the high dose vaccine in a fetal challenge model will follow.
Animals↗
Adhesion induced expression of the serine/threonine kinase Fnk in human macrophages.
Members of the polo subfamily of protein kinases play crucial roles in cell proliferation. To study the function of this family in more detail, we isolated the cDNA of human Fnk (FGF-inducible kinase) which codes for a serine/threonine kinase of 646 aa. Despite the homology to the proliferation-associated polo-like kinase (Plk), tissue distribution of Fnk transcripts and expression kinetics differed clearly. In contrast to Plk no correlation between cell proliferation and Fnk gene expression was found. Instead high levels of Fnk mRNA were detectable in blood cells undergoing adhesion. The transition of monocytes from peripheral blood to matrix bound macrophages was accompanied by increasing levels of Fnk with time in culture. Neither treatment of monocytes with inducers of differentiation nor withdrawal of serum did influence Fnk mRNA levels significantly, suggesting that cell attachment triggers the onset of Fnk gene transcription. The idea that Fnk is part of the signalling network controlling cellular adhesion was supported by the analysis of the cytoplasmic distribution of the Fnk protein and the influence of its overexpression on the cellular architecture. Fnk as fusion protein with GFP localized at the cellular membrane in COS cells. Dysregulated Fnk gene expression disrupted the cellular f-actin network and induced a spherical morphology. Furthermore, Fnk binds to the Ca2+/integrin-binding protein Cib in two-hybrid-analyses and co-immunoprecipitation in assays. Moreover, both proteins were shown to co-localize in mammalian cells. The homology of Cib with calmodulin and with calcineurin B suggests that Cib might be a regulatory subunit of polo-like kinases.
Actins↗
Identification and functional characterization of the human and murine fibroblast growth factor receptor 4 promoters.
Fibroblast growth factor receptors (FGFRs) play crucial roles in signal transduction of adult tissues and during embryonic development. To study the transcriptional control, we isolated and characterized the promoter of human FGFR4. Two transcription initiation sites were identified. The deletion analysis in different cell types defined a core promoter reaching from -9 to -198, lacking TATA and CCAAT boxes but displaying high GC content (77%) in a stretch of 300 bp upstream of the major mRNA start. This region harbors multiple binding motifs for transcription factors. Moreover, the region between -1085 and -1140 contains a potential repressor element, which downregulates transcriptional activity. To identify conserved regulatory elements, we isolated and analyzed also the murine FGFR4 promoter. Only one transcription start was identified using RNase protection assays. Sequence alignment of human and mouse shows a striking similarity in the core promoter region of both genes, encompassing conserved transcription factor binding sites and a splice acceptor site. Furthermore, the region containing the putative repressor element is also conserved suggesting a functional role for gene expression.
Animals↗
Acute renal failure associated with rofecoxib.
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Acute Kidney Injury↗
Nitrosative stress in primary glial cultures after induction of the inducible isoform of nitric oxide synthase (i-NOS).
Primary glial cultures are able to express the inducible isoform of nitric oxide synthase (i-NOS) upon stimulation by bacterial lipopolysaccharides (LPS) and gamma-interferon (gamma-IfN). Immunocytochemical studies revealed, that under our experimental conditions i-NOS is expressed exclusively by the microglial cells and not in the astrocytes. Nitric oxide (NO) formation represents an oxidative load for the microglial cells, as observed by the oxidation rate of the ROS- and peroxynitrite indicator dichloro-dihydrofluorescein (DCF-H) in these cells. However, cell viability was not affected by the nitric oxide formation, indicating some form of protection against the higher oxidative load. Upregulation of Mn-SOD in the mitochondria in the course of the induction of i-NOS and, compared to the astrocytes, higher GSH levels in the microglial cells probably explain the resistance of the cultures against nitrosative stress. Increased SOD-activities in the mitochondria could lower the superoxide concentration in this organelle and may prevent an oxidative and/or nitrosative damage via a decreased peroxynitrite formation. The higher GSH-levels in the microglial cells of unstimulated cultures represents a buffer which, under the conditions of i-NOS catalyzed NO-formation, prevents a decline of the microglial GSH-levels below that of the astrocytes.
Animals↗
Differentially displayed genes in neuroblastoma cells treated with a mitochondrial toxin: evidence for possible involvement of ICAM-1 in 3-nitropropionic acid-mediated neurodegeneration.
The mitochondrial toxin 3-nitropropionic acid (3-NPA) causes neurodegeneration in the basal ganglia and neurological symptoms resembling Huntington's disease (HD) when applied to primates or rodents, and therefore might be used as an animal model for this disorder. For that reason, the molecular mechanisms involved in 3-NPA-induced neurodegeneration are of considerable interest. In our model, murine neuroblastoma cells (Neuro-2a) were treated with different doses of 3-NPA, and changes in gene expression were analyzed by means of mRNA differential display (DDRT-PCR). Using 18 primer combinations, we have identified a set of 33 candidate cDNAs deriving from 29 excised DDRT bands whose expression appeared to be changed in response to the 3-NPA insult (mostly elevated). DNA sequencing revealed that novel, as well as previously described genes, are included in this panel. Amongst the known cDNAs, the differential mRNA expression of the ribosomal proteins S6 and L40, of the protein kinase A (PKA) catalytic beta subunit and of the intercellular adhesion molecule ICAM-1 could be verified using Northern hybridization and RT-PCR, respectively. Furthermore, ICAM-1 expression could also be shown to increase at the protein level, which points to a possible function for this molecule in neuronal cells in the course of neurodegeneration. The results may prove useful in elucidating the multiple processes causing neurodegeneration subsequent to lesions by mitochondrial toxins and excitotoxins as well.
Animals↗
Distribution, targeting, and internalization of the sst4 somatostatin receptor in rat brain.
Somatostatin mediates its diverse physiological effects through a family of five G-protein-coupled receptors (sst(1)-sst(5)); however, knowledge about the distribution of individual somatostatin receptor proteins in mammalian brain is incomplete. In the present study, we have examined the regional and subcellular distribution of the somatostatin receptor sst(4) in the rat CNS by raising anti-peptide antisera to the C-terminal tail of sst(4). The specificity of affinity-purified antibodies was demonstrated using immunofluorescent staining of HEK 293 cells stably transfected with an epitope-tagged sst(4) receptor. In Western blotting, the antiserum reacted specifically with a broad band in rat brain, which migrated at approximately 70 kDa before and approximately 50 kDa after enzymatic deglycosylation. sst(4)-Like immunoreactivity was most prominent in many forebrain regions, including the cerebral cortex, hippocampus, striatum, amygdala, and hypothalamus. Analysis at the electron microscopic level revealed that sst(4)-expressing neurons target this receptor preferentially to their somatodendritic domain. Like the sst(2A) receptor, sst(4)-immunoreactive dendrites were often closely apposed by somatostatin-14-containing fibers and terminals. However, unlike the sst(2A) receptor, sst(4) was not internalized in response to intracerebroventricular administration of somatostatin-14. After percussion trauma of the cortex, neuronal sst(4) receptors progressively declined at the sites of damage. This decline coincided with an induction of sst(4) expression in cells with a glial-like morphology. Together, this study provides the first description of the distribution of immunoreactive sst(4) receptor proteins in rat brain. We show that sst(4) is strictly somatodendritic and most likely functions in a postsynaptic manner. In addition, the sst(4) receptor may have a previously unappreciated function during the neuronal degeneration-regeneration process.
Age Factors↗
Identification of alpha3, alpha4, and alpha5 chains of type IV collagen as alloantigens for Alport posttransplant anti-glomerular basement membrane antibodies.
BACKGROUND: Alport syndrome is a hereditary disorder of basement membranes especially affecting the kidneys, ears, and eyes. Some patients who undergo renal transplantation lose their kidneys as a result of posttransplant anti-glomerular basement membrane (anti-GBM) disease. METHODS: In the present study, we analyzed serum from 21 unselected Alport patients who underwent renal transplantation. Eleven samples were from patients without posttransplant anti-GBM nephritis, and 10 were from patients with this disease. RESULTS: Thirteen serum samples [10 alport posttransplant nephritis serum (APTN) and three Alport posttransplant serum (APT)] revealed linear binding to the GBM by indirect immunofluorescence. By using direct ELISA and immunoblotting with GBM constituents and type IV collagen NC1 domains from bovine, human, and recombinant sources, we detected anti-GBM antibodies in all Alport patients in varying titers. Five samples showed specific reactivity to the alpha3 chain, four to the alpha5 chain, six to both alpha3 and alpha5 chains, one to the alpha3 and alpha4 chains, and two to the alpha3, alpha4, and alpha5 chains of type IV collagen. The varied spectrum of reactivities was present equally in nephritic and non-nephritic sera. Ten control samples from non-Alport transplant patients did not exhibit specific binding to the GBM. CONCLUSIONS: These results suggest that the absence of alpha3, alpha4, and alpha5 chains of type IV collagen in the Alport kidney leads to alloantibodies in all Alport patients who receive transplants, irrespective of whether they develop nephritis or not. Although all Alport transplant patients develop this humoral response, only a select few develop anti-GBM disease. We suggest that this difference could be attributable to a genotypic effect on the ability of some individuals to launch a cell-mediated immune response.
Animals↗
Cellular distribution of superoxide dismutases in the rat CNS.
Superoxide dismutase (SOD) is considered to be a major factor in protection of nervous tissue against excitotoxic and ischemic/hypoxic lesion. Controversial reports about the localization of SOD after such an insult prompted us to re-investigate immunocytochemically the localization of the enzyme in the brain and spinal cord using specific antibodies against the manganese (Mn-SOD) and copper/zinc (Cu/Zn-SOD) containing isoenzyme in combination with cell type specific markers. CNS tissue sections were analyzed by confocal laser scanning microscopy and digital photo imaging. Cu/Zn-SOD immunoreactivity was found to be located predominantly in astrocytes throughout the CNS. The staining was found in the cytoplasm, in cellular processes and, less intensive, in the nucleus sparing the nucleolus. At a lower level the enzyme was also detectable in neuronal perikarya and in structures of the neuropil. Motoneurons of the spinal cord displayed an enhanced Cu/Zn-SOD staining intensity, when compared to brain neurons. In contrast the Mn-containing isoenzyme was predominantly localized to neurons and their processes throughout the brain and the spinal cord. Confirming the mitochondrial localization of the enzyme, a granular staining pattern sparing the nucleus was observed. Mn-SOD stained mitochondria were also seen in astroglial cells but the staining intensity was, on the whole, much lower compared to neurons, and often hardly detectable. It seems reasonable to conclude that differences in the basal content of SOD-isoenzymes may contribute to different cellular susceptibilities in neurodegenerative processes that are accompanied by oxidative stress.
Animals↗
Induction of nitric oxide synthesis lowers intracellular glutathione in microglia of primary glial cultures.
Glutathione (GSH) is one of the most important antioxidants involved in detoxification of reactive oxygen and nitrogen species. We investigated the changes in intracellular GSH in primary glial cultures stimulated to produce inducible nitric oxide synthase and subsequently nitric oxide by bacterial lipopolysaccharide and gamma interferon treatment. Intracellular GSH content was measured by both the monochlorobimane fluorescence microscopy method and the GSH reductase recycling assay (Tietze. Anal Biochem 27:502-522, 1969.). Our results show that irrespective of the assay used the GSH content in stimulated cultures decreased to almost half that of control cultures. This decrease in GSH content was accompanied by an increase in S-nitrosoglutathione in the stimulated cultures. Analysis of the GSH related fluorescence images showed that the fluorescence intensity was lowered exclusively in microglial cells whereas that of astrocytes remained almost unchanged. The present study in conjunction with our previous investigation (Chatterjee et al. Glia 27:152-161, 1999) can be interpreted to imply that the higher GSH levels in untreated microglia are a mechanism to withstand nitric oxide synthase induced oxidative and nitrosative stress and therefore the GSH levels in microglia drop to astrocyte levels after induction.
Animals↗
Telepathology by the Internet.
A new concept for telemicroscopy has recently been introduced using the Internet and conventional web browser, with Java support for microscope remote control as well as image transfer and discussion (http://amba.charite.de/telemic/). The system has two major components: the telemicroscopy server, which is a computer with Internet access connected to the automatic microscope, and the telemicroscopy client, who remotely operates the microscope. This simplified telemicroscopy system allows any Internet user to become a consultant for telepathology without the acquisition of specialized hardware or software. For the inquirer seeking advice, however, this solution is still very expensive, since it requires a fully automated microscope. The present study describes a system that can be used for conventional microscopes. A video camera mounted on a microscope with a photo tube is connected to the frame grabber of a PC. Java-based telemicroscopy software transforms the computer into an Internet server, which automatically distributes new microscope images, after manual operations, to all connected clients. Any Internet user can access the web page of the server to become a telemicroscopy client. A Chat function allows for the online exchange of written text and a Discuss function enables the mouse button to display an arrow to all connected clients, which highlights distinct structures of the images. The system was optimized for simplicity, while presenting all features that are necessary to show and discuss difficult cases with any expert in the field who has Internet access. It offers new perspectives for telepathology and it is envisaged that many pathologists and scientists will use this facility to connect their personal microscopes to the Internet, forming a network for teleconsultation. To foster this development, the software described in this paper is being made freely available. Hopefully, this development will promote communication between pathologists and may thus increase the quality of diagnosis. Information on inquiry and installation of the software is available at the website mentioned above. Telemicroscopy sessions using the Telemic version for conventional microscopes can be scheduled by contacting the authors by e-mail (iver. petersen@charite.de).
Communication↗