Biomedical subjects
Publications and source records attributed to G Thiel.
Because of the severe shortage of cadaver organs, the transplantation of living donated organs from related and also from unrelated donors has been suggested; graft survival is similar or better than with cadaver kidneys. Therefore, kidneys from living donors are a large and valuable source for transplantation which help increase the pool of organs and thus decrease the time spent waiting for organ transplantation.
Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis. The 5'-flanking region of the human synaptobrevin II gene is very (G+C)-rich and contains a 13-bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr-1. To test whether Sp1 and zif268/egr-1 interact with this motif, gel retardation assays were performed. These assays revealed that both transcription factors bind to the (G+C)-rich motif of the synaptobrevin II gene in vitro. The binding of Sp1 was additionally confirmed by supershift analysis with antibodies specific for Sp1. To determine whether zif268/egr-1 plays a role in controlling synaptobrevin II gene expression, a plasmid was constructed containing the (G+C)-rich motif of the synaptobrevin II gene upstream of a minimal promoter and the Escherichia coli chloramphenicol acetyltransferase (CAT) gene as a reporter. This plasmid was transfected into CHO-K1 cells together with an expression vector encoding zif268/egr-1. Zif268/egr-1 failed to activate transcription from this reporter gene, although it transactivated a reporter gene containing an identical (G+C)-rich motif derived from the human synapsin I promoter. Overexpression of Sp1, however, clearly activated transcription of a reporter gene under the control of the synaptobrevin II promoter (G+C)-rich sequence in Drosophila SL2 cells, which provided an Sp1-deficient background. Furthermore, a glutathione S-transferase protein containing the DNA-binding domain of Sp1 was shown to function as a dominant negative form of Sp1, reducing transcription of the synaptobrevin II promoter-CAT reporter gene in mammalian cells to basal levels. From these data, we conclude that the zif268/egr-1-binding site in the synaptobrevin II promoter is not functionally active. Instead, an overlapping Sp1-binding site in this (G+C)-rich region clearly mediates constitutive transcriptional activation.
The enzyme fructose-1,6-bisphosphate aldolase consists of three isozymes that are expressed in a tissue-specific manner. Using antibodies against aldolase B and C, it is shown that aldolase C is expressed in virtually all neuronal cell lines derived from the central and peripheral nervous system. Recently, experiments with transgenic mice indicated that a (G+C)-rich region of the aldolase C promoter might function as a neuron-specific control element of the rat aldolase C gene [Thomas, M., Makeh, I., Briand, P., Kahn, A. & Skala, H. (1993) Eur. J. Biochem. 218, 143-151). To functionally analyse this element, a plasmid consisting of four copies of this (G+C)-rich sequence, a TATA box, and the rabbit beta-globin gene as reporter was constructed. This plasmid was transfected into neuronal and nonneuronal cell lines and transcription was monitored by RNase protection mapping of the beta-globin mRNA. It is shown that the (G+C)-rich element of the aldolase C promoter directs transcription in neuronal as well as in nonneuronal cells. In contrast, the synapsin I promoter, used as a control for neuron-specific gene expression, directed transcription only in neuronal cells. In gel-retardation assays, two major DNA-protein complexes were detected with the (G+C)-rich element of the aldolase C promoter used as a DNA probe and nuclear extracts from brain and liver as a source for DNA-binding proteins. These DNA-proteins interactions could be impaired by a DNA probe that contained an Sp1-binding site, indicating that Sp1 or an Sp1-related factor binds to the aldolase C promoter (G+C)-rich element. This was confirmed by supershift analysis with antibodies specific for Sp1. The zinc finger transcription factor zif268/egr-1, also known to recognize a (G+C)-rich consensus site, did not, however, bind to the (G+C)-rich motif of the aldolase C promoter, nor could it stimulate transcription in transactivation assays from this control region. From these data, we conclude that the (G+C)-rich element of the aldolase C promoter functions as a constitutive transcriptional response element mediated by Sp1 and Sp1-related transcription factors.
Explore the source record for details and available documents.
Secretogranin II is an acidic secretory protein with a widespread distribution in secretory granules of neuronal and endocrine cells. The secretogranin II gene contains, like other members of the granin family, a cAMP response element (CRE) in its upstream region. To investigate the functional significance of this motif, intracellular cAMP levels were increased in a neuronal cell line derived from the septal region of the brain and the level of secretogranin II gene expression was analysed. It was found that increased cAMP levels did, in fact, induce secretogranin II gene expression. To analyse the cis-acting sequence responsible for this induction, a hybrid gene containing the upstream region of the mouse secretogranin II gene fused to beta-globin as a reporter was constructed. Transfection analysis revealed that cAMP-induced transcription of the secretogranin II promoter/beta-globin gene in septal and insulinoma cells. DNA-protein binding assays showed that recombinant CRE-binding protein (CREB), produced in bacteria or human cells, bound in a sequence-specific manner to the secretogranin II promoter CRE. Moreover, deletion mutagenesis revealed that the CRE motif is a bifunctional genetic regulatory element in that it mediates basal as well as cAMP-stimulated transcription. Interestingly, cAMP had no effect upon secretogranin II gene transcription in PC12 and neuroblastoma cells. An increase in the intracellular cAMP concentration activated a GAL4-CREB fusion protein upon transcription in neuroblastoma cells indicating the integrity of the cAMP signaling pathway to the nucleus. Basal as well as cAMP-stimulated transcription, directed from the secretogranin II promoter was, however, impaired in insulinoma cells by overexpression of CREB-2, a negative-acting CRE-binding protein. These results indicate that competitive effects are likely to occur between CRE-bound transcriptional activators and repressors. We conclude that cAMP-stimulated induction of secretogranin II gene transcription is mediated by the CRE motif in a cell-type-specific manner, and is likely to depend on the balance between positive and negative CRE-binding proteins in a particular cell type.
We have constructed a vector, pHIVTATA-CAT, that contains the Escherichia coli cat gene, encoding chloramphenicol acetyltransferase, under the control of a minimal promoter consisting of the HIV TATA box and the adenovirus major late promoter initiator element. Putative transcriptional elements can be inserted either directly upstream from the TATA box or downstream from the reporter gene in an enhancer position. Transcription can be monitored enzymatically or by RNase protection mapping. An analysis of mRNAs generated from pHIVTATA-CAT constructs revealed that transcription starts at the transcription start point and no read-through transcripts are generated.
The synapsins are a family of neuron-specific phosphoproteins that selectively bind to small synaptic vesicles in the presynaptic nerve terminal. The human synapsin I gene was functionally analyzed to identify control elements directing the neuron-specific expression of synapsin I. By directly measuring the mRNA transcripts of a reporter gene, we demonstrate that the proximal region of the synapsin I promoter is sufficient for directing neuron-specific gene expression. This proximal region is highly conserved between mouse and human. Deletion of a putative binding site for the zinc finger protein, neuron-restrictive silencer factor/RE-1 silencing transcription factor (NRSF/REST), abolished neuron-specific expression of the reporter gene almost entirely, allowing constitutively acting elements of the promoter to direct expression in a non-tissue-specific manner. These constitutive transcriptional elements are present as a bipartite enhancer, consisting of the region upstream (nucleotides -422 to -235) and downstream (nucleotides -199 to -143) of the putative NRSF/REST-binding site. The latter contains a motif identical to the cAMP response element. Both regions are not active or are only weakly active in promoting transcription on their own and show no tissue-specific preference. From these data we conclude that neuron-specific expression of synapsin I is accomplished by a negative regulatory mechanism via the NRSF/REST binding motif.
Consistent tumor-specific chromosomal aberrations have not been described in low-grade astrocytic tumors. The most frequent genetic alterations are mutations of the TP53 tumor suppressor gene and/or loss of heterozygosity (LOH) on 17p that occur in about 30% of the cases in adult patients but that are uncommon in childhood tumors. We used comparative genomic hybridization (CGH) to map DNA copy number alterations in 18 primary low-grade astrocytic tumors (ten adult patients and eight children). A gain of chromosome arm 7q was the most frequent event detected in five of ten astrocytomas (50%) from adult patients, followed by DNA amplification on chromosome arm 8q and gain on 12p (two cases). Loss of chromosomal regions on 1p, 4q, and the X chromosome was observed in two of ten cases [including one patient afflicted with Turner syndrome (45,X)]. In contrast, no consistent changes were observed in low-grade astrocytomas in children. A loss of the X chromosome was the sole aberration detected in two of eight cases using DNA extracted from the normal brain tissue. The findings suggest that a gain of 7q is an early event in the initiation of astrocytomas in adult patients. The discrepant findings in low-grade astrocytic tumors in adults compared to tumors in children support the the hypothesis that there might be different mechanisms responsible for tumor development.
The ESOT Council conducted an inquiry on new immunosuppressive substances in order to help keep members of ESOT informed. Thirty-one pharmaceutical companies were sent a questionnaire indicating whether they were developing new immunosuppressive substances/antibodies. Sixteen companies responded: 11 furnished information on 16 substances; 5 said that they were not developing any new immunosuppressive agents. Fifteen companies did not reply at all. The results of the first inquiry are reported here.
Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles. Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex. These currents are essentially mediated by K+ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage and inhibited by extracellular Ca2+ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca(2+)-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive states via independent first order reactions: a fast inactivation/activation by Ca(2+)-binding and -release, respectively (rate constants >> 10(3) sec-1) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the range of 10(3) sec-1). With 10 mM K+ and 1 mM Ca2+ in the external solution, intact coleoptile cells have a membrane voltage (V) of -105 +/- 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K+ uptake required for cell elongation. Extracellular TEA or Ca2+, which inhibit the K+ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects of Ca2+ and TEA on both processes and the similar Ca2+ concentration required for half maximal inhibition of growth (4.3 mM Ca2+) and for conductance (1.2 mM Ca2+) suggest that K+ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles.
Explore the source record for details and available documents.
OBJECTIVES: Incidence and risk factors of post-transplant monoclonal gammopathy were studied in renal transplant patients who received their grafts between 1982 and 1992 (n = 390 grafts). Immunoelectrophoresis was performed at annual intervals after transplantation. RESULTS: Forty-six cases of clonal gammopathy were detected: 35 monoclonal, 11 bi- or triclonal, with a predominance of IgG and kappa light-chain subtypes (IgG, 39; IgA, 3; IgM, 4; kappa, 35; lambda, 19). Gammopathy was incidence of gammopathy was 10.7%, much higher than expected for a group of similar age from the general population. Thirty of the 46 gammopathies appeared within the first 2 years of transplantation. Gammopathy never progressed to multiple myeloma during follow-up (median 1 year; (range 0-10)); one patient subsequently developed Kaposi sarcoma. The 2-year incidence of gammopathy was much higher in patients transplanted in 1989-1991 (23/142) than in 1982-1988 (7/248) (P < 0.0001). This coincided with the use of quadruple induction immunosuppression (cyclosporin A+azathioprine+prednisone plus either ATG-fresenius (ATG-F) or OKT3) since 1989. The risk for acquiring gammopathy within 2 years of transplantation was 14.7% (95% CI 9.2, 20.3%) in patients receiving quadruple induction therapy, but only 3.0% (CI 1.2, 6.1%) without such therapy (P < 0.0001). The risk for patients receiving quadruple immunosuppression with OKT3 was 24.5%, significantly greater than with ATG-F (11.8%, P < 0.05). Discriminant analysis revealed that the type of immunosuppression, but not age or year of transplantation, were independent risk factors for gammopathy. CONCLUSION: Monoclonal gammopathy frequently occurs after renal transplantation. Risks are higher for patients receiving quadruple induction immunosuppression, particularly if it includes OKT3. Follow-up of these patients is warranted for the early detection of malignant transformation.
Explore the source record for details and available documents.
BACKGROUND: Hyperuricaemia is a well known side-effect of cyclosporin A (CsA) treatment. The pathogenic mechanisms, however, remain controversial. There is no convincing evidence that hyperuricaemia is due to CsA-induced, impaired tubular handling of uric acid. The impact of diminished GFR in this particular context has never been investigated. METHODS: We prospectively studied plasma uric acid, inulin clearances, and fractional clearances of uric acid in two groups of CsA-treated patients (bone-marrow transplant patients, n = 50; renal transplant patients, n = 32), and one healthy control group without CsA (living related kidney donors, n = 28). Bone-marrow transplant patients were examined before transplantation and 6, 12, 18, 24 months after transplantation, renal transplant patients 1 year after transplantation, and living related kidney donors before and 1 year after unilateral nephrectomy. RESULTS: After 1 year of CsA treatment, hyperuricaemia was found in 36% of bone-marrow transplant patients and in 53% of renal transplant patients. Thirty per cent of living related kidney donors were borderline hyperuricaemic 1 year after unilateral nephrectomy. The fractional clearance of uric acid, measured serially in bone-marrow transplant patients did not change significantly over time; it was, however, slightly higher during CsA treatment than after CsA withdrawal. Moreover, the bone-marrow transplant patients' fractional clearance of uric acid was not statistically different from the renal transplant patients' and the living related kidney donors' (values 1 year after transplantation/unilateral nephrectomy: bone-marrow transplant patients, 15.3 +/- 2.3%; renal transplant patients, 11.9 +/- 0.9%; living related kidney donors, 11.1 +/- 0.8%). The GFR at 1 year measured by inulin clearance, was identical in the CsA-treated groups and slightly higher in the living related kidney donors (bone-marrow transplant patients, 51 +/- 6 ml/min per 1.73 m2; renal transplant patients, 49 +/- 3 ml/min per 1.73 m2; living related kidney donors, 61 +/- 2 ml/min per 1.73 m2). CONCLUSION: There is no evidence for impaired tubular handling of uric acid, induced by a CsA-specific tubulotoxic effect. Hyperuricaemia in CsA-treated transplant patients can therefore be attributed to the cyclosporin-associated decrease of GFR.
Synapsin II is a neuron-specific phosphoprotein that selectively binds to small synaptic vesicles in the presynaptic nerve terminal. Here we report the cloning and sequencing of the 5'-flanking region of the human synapsin II gene. This sequence is very GC-rich and lacks a TATA or CAAT box. Two major transcriptional start sites were mapped. A hybrid gene consisting of the Escherichia coli chloramphenicol acetyltransferase gene under the control of 837 base pairs of the synapsin II 5'-upstream region was transfected into neuronal and nonneuronal cells. While reporter gene expression was low in neuroblastoma and non-neuronal cells, high chloramphenicol acetyltransferase activities were monitored in PC12 pheochromocytoma cells. However, there was no correlation between reporter gene expression in the transfected cells and endogenous synapsin II immunoreactivity. Using DNA-protein binding assays we showed that the transcription factors zif268/egr-1, polyoma enhancer activator 3 (PEA3), and AP2 specifically contact the synapsin II promoter DNA in vitro. Moreover, the zif268/egr-1 protein as well as PEA3 were shown to stimulate transcription of a reporter gene containing synapsin II promoter sequences. In the nervous system, zif268/egr-1 functions as a "third messenger" with a potential role in synaptic plasticity. PEA3 is expressed in the brain and its activity is regulated by proteins encoded from non-nuclear oncogenes. We postulate that zif268/egr-1 and PEA3 couple extracellular signals to long-term responses by regulating synapsin II gene expression.
Explore the source record for details and available documents.
Osteopenia has been observed to occur frequently after renal transplantation. The present study was undertaken to assess whether an immunosuppressive regimen combining cyclosporine with no or the lowest possible maintenance doses of glucocorticoid may prevent osteopenia after kidney transplantation. Thirty-four patients were prospectively followed for two years. Serial blood drawings were done for determination of serum indices of calcium and bone metabolism and an iliac crest bone biopsy was performed at time of transplantation. A second bone biopsy was done in 20 patients during the second year of observation. Creatinine clearance was 56 +/- 6 ml/min one year and 46 +/- 6 ml/min two years after transplantation. Serum parathyroid hormone levels were elevated in 24 patients at time of grafting, decreased significantly thereafter, but remained above the normal range. Ten patients had low or normal serum parathyroid hormone levels at time of transplantation and showed a significant increase after grafting. Two years after transplantation, the mean cumulative dose of prednisone was 5.9 +/- 0.5 g. After the first six months, 30-40% of the patients were not on maintenance doses of steroids. None of the patients experienced fractures, and cancellous bone volume was within or above the normal range in all repeat bone biopsies. It is of note that metabolic bone abnormalities did not resolve 1-2 years after transplantation despite normalization of serum 1,25 vitamin D levels. The histologic abnormalities at the time were consistent with the bone findings in renal failure suggesting resistance of bone to normal circulating levels of 1,25 vitamin D.
We carried out a randomized prospective trial to compare OKT3 (5 mg/d, 51 patients) with ATG-Fresenius (ATG-F, 4 mg/kg/d, 53 patients) for induction therapy after renal transplantation, concerning side effects, rejection, and infection incidence within a one year follow-up period. Concomitant immunosuppression included azathioprine/steroids from day 0 and cyclosporine A from day 4. OKT3 patients experienced significantly more and more-severe side effects, particularly pyrexia, headache, and pulmonary fluid overload. One-year graft survival was excellent in the ATG-F group (91%), but only 78% in the OKT3 group (P < 0.05) due to a series of rejections that occurred beyond day 100; patient survival (96% and 92%) was similar in both groups. OKT3-treated patients experienced more biopsy-proven rejections (0.6 +/- 0.1/pt.) than ATG-F patients (0.3 +/- 0.1, P < 0.05), and there was a similar, albeit not significant trend in clinical rejections (OKT3: 1.1 +/- 0.2/pt.; ATG-F: 0.8 +/- 0.1/pt.). Infections were more common in the OKT3 group (OKT3: 3.2 +/- 0.3, ATG-F: 2.0 +/- 0.2, P < 0.05), although this was entirely attributable to "minor" infections. On days 1 through 6, CD3 counts were more profoundly depressed with OKT3 therapy. Beyond day 10, however, CD3 counts were lower in the ATG-F group, as were CD2 counts, CD4 counts, and the CD4/CD8 ratio, suggesting a more prolonged immunosuppressive effect of ATG-F. Sensitization occurred more frequently with OKT3 (31%) than with ATG-F (10%), but was usually irrelevant, except in two patients (one in each group), whose grafts were lost because of immunization against OKT3 and ATG-F, respectively. In conclusion, a 7-day induction therapy with OKT3 does not improve outcome or diminish immunological graft loss when compared with ATG-F, but is associated with more rejections, infections, and side effects. ATG-F appears to be preferable for induction immunosuppression after renal transplantation.