Biomedical subjects
F Zimmermann
Publications and source records attributed to F Zimmermann.
Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice.
The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.
Locally advanced rectal cancer: resection and intraoperative radiotherapy using the flab method combined with preoperative or postoperative radiochemotherapy.
PURPOSE: Surgery often fails to achieve local control in advanced rectal cancer. Additional measures are necessary to prevent local recurrence. The aim of this study was to evaluate intraoperative radiation therapy (IORT) (flab technique) combined with preoperative or postoperative radiochemotherapy. PATIENTS/METHODS: IORT is performed using a flexible flab containing hollow plastic tubes that are connected to a multichannel afterloading device with a 370 Gbq-192-Ir source. Patients receive an intraoperative dose of 15 Gy. Target volumes were measured in a cadaver experiment. From 1989 to 1993, 38 patients were included in this study. Nineteen patients were staged as T3 tumors by preoperative endosonography (Group I) and 19 as T4 tumors (Group II). Patients in Group I underwent resection (abdominoperineal resection (APR), 16; anterior resection, 3) and IORT, followed by postoperative radiochemotherapy (50 Gy/5-fluorouracil), whereas patients in Group II received preoperative radiochemotherapy (40 Gy/5-fluorouracil) followed by resection (APR, 18; anterior resection, 1) and IORT. Mean follow-up was 25.5 months. RESULTS: Operative radicality in Group I was RO (13), R1 (3), and R2 (3), and in Group II it was RO (14), R1 (3), R2 (2). R2 resections were attributable to preoperative undetected distant metastases. Perioperative mortality was 0 percent in Group I and 10.5 percent (n = 2) in Group II. Postoperative morbidity was 53 percent (n = 10) in Group I and 84 percent (n = 16) in Group II with delayed sacral wound healing being the predominant problem. Stenosis of the ureter occurred in two patients (Group II). Late or persistent therapy-related complications were seen in two patients in Group I and in six patients in Group II. Local recurrence developed in three patients in Group I (15.8 percent) and in two patients in Group II (10.5 percent). Survival data do not reach statistical significance between the two groups because of small numbers but show a favorable trend for the preoperative radiochemotherapy group. When compared with a matched historical control group of patients receiving resection only, adjuvant/neoadjuvant radiotherapy with resection/IORT improves survival significantly. CONCLUSION: The flab method is a simple but especially practical technique for IORT in the pelvis. Adjuvant/neoadjuvant therapy combined with resection/IORT is associated with high morbidity but acceptable mortality. Preliminary survival data are encouraging and call for a controlled prospective randomized trial.
The sensitivity of in vitro erythropoietic progenitor cells to different erythropoietin reagents during development and the role of cell death in culture.
With the availability of several recombinant erythropoietin (Epo) reagents, it has been possible to undertake a systematic study of the relative Epo sensitivity of late erythroid colony-forming units (CFU-E) in 8.5-day embryos, 13.5-day fetal liver, and adult bone marrow of the mouse. All Epo preparations tested, including one from impure sheep and a highly purified human native Epo preparation, produced parallel, but displaced, dose-response curves when Epo concentration was plotted against percent CFU-E response calculated from the optimal Epo concentration. It was found that the CFU-E derived from 8.5-day embryos demonstrated the greatest Epo sensitivity which decreased in fetal liver and adult bone marrow CFU-E populations. Modifications to the culture system allowed CFU-E to be stimulated with as little as 0.003 mU/mL, equivalent to approximately 0.03 fg Epo. Under these culture conditions, no evidence for apoptosis was found, although a normal programmed cell death function cannot be ruled out.
Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice.
The arginine residue at position 586 of the GluR-B subunit renders heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive glutamate receptor channels impermeable to calcium. The codon for this arginine is introduced at the precursor messenger RNA (pre-mRNA) stage by site-selective adenosine editing of a glutamine codon. Heterozygous mice engineered by gene targeting to harbor an editing-incompetent GluR-B allele synthesized unedited GluR-B subunits and, in principal neurons and interneurons, expressed AMPA receptors with increased calcium permeability. These mice developed seizures and died by 3 weeks of age, showing that GluR-B pre-mRNA editing is essential for brain function.
Fast beam-ion instability. I. Linear theory and simulations.
Explore the source record for details and available documents.
Fast beam-ion instability. II. Effect of ion decoherence.
Explore the source record for details and available documents.
The initiation of the hemopoietic system. The response of embryonic cells to growth factors and expression of erythropoietin and erythroid-relevant genes during murine development.
Explore the source record for details and available documents.
Ontogenetic regulation of mouse Ren-2d renin gene in transgenic hypertensive rats, TGR(mREN2)27.
TGR(mREN2)27 is a new monogenetic rat model with fulminant hypertension, low kidney renin, and high extrarenal renin gene expression. This study characterizes and compares expression of the Ren-2 gene in TGR(mREN2)27 with that in DBA/2 mice and with renin gene expression in rats. Except in the submandibular gland, the tissue-specific expression of Ren-2 is similar in TGR(mREN2)27 and DBA/2. This demonstrates maintenance of tissue specificity. Organs that are involved in cardiovascular regulation, such as the adrenal gland, kidney, and brain, express the Ren-2 gene before hypertension has developed, consistent with the possibility of a causal relationship between transgene expression in these tissues and hypertension. Because these tissues express the renin gene in nontransgenic rats as well, we suggest that this model can be used to study the regulation of renin gene expression and its role in hypertension at these sites. In addition, as an indication that interactions may exist between blood pressure and renin gene expression, we describe reciprocal changes in blood pressure and Ren-2 mRNA levels in the kidney and brain.
Selective generation of antigen-specific human hybridomas optimized for large scale growth in serum-free medium.
The direct propagation of newly formed human hybridomas in serum-free medium selects for hybrids with a metabolism best suited to growth in this environment. Under optimal culture conditions, this procedure results in the generation of antigen-specific human hybridomas comparable in frequency, stability, and antibody secretion rate to that obtained with murine hybridomas. After a transient phase of a few days in the appropriate selection medium supplemented with 1% serum, hybridomas grow in serum-free medium in stationary cultures with a cell doubling time of 15-25 h and an antibody production rate averaging 12 micrograms/10(6) cells/day. Clones propagated in bioreactors exhibited a cell doubling time of 29-35 h and an antibody secretion rate of 10-21 micrograms/10(6) cells/day.
Species specificity of renin kinetics in transgenic rats harboring the human renin and angiotensinogen genes.
The renin-angiotensin system (RAS) is the most important regulatory system of electrolyte homeostasis and blood pressure. We report here the development of transgenic rats carrying the human angiotensinogen TGR-(hAOGEN) and human renin TGR(hREN) genes. The plasma levels and tissue distribution of the transcription and translation products from both genes are described. A unique species specificity of the enzyme kinetics was observed. The human RAS components in the transgenic rats did not interact with the endogenous rat RAS in vivo. Instead, infusions of exogenous human RAS components specifically interacted with human transgene translation products. Thus, infusion of human renin in TGR(hAOGEN) led to an increase of angiotensin II and an elevation of blood pressure, which could not be antagonized by the human-specific renin enzyme inhibitor Ro 42-5892. Rat renin also elevated blood pressure and angiotensin II in TGR(hAOGEN); however, this effect was not antagonized by the human renin inhibitor. Compared to mice, rats offer the advantage of chronic instrumentation and repetitive, sophisticated, hemodynamic, and endocrinological investigations. Thus, transgenic rat models with human-specific enzyme kinetics permit primate-specific analyses in non-primate in vivo and in vitro experimental systems.
High blood pressure in transgenic mice carrying the rat angiotensinogen gene.
Transgenic mice were generated by injecting the entire rat angiotensinogen gene into the germline of NMRI mice. The resulting transgenic animals were characterized with respect to hemodynamics, parameters of the renin angiotension system, and expression of the transgene. The transgenic line TGM(rAOGEN)123 developed hypertension with a mean arterial blood pressure of 158 mmHg in males and 132 mmHg in females. In contrast, the transgenic line TGM(rAOGEN)92 was not hypertensive. Rat angiotensinogen was detectable only in plasma of animals of line 123. Total plasma angiotensinogen and plasma angiotensin II concentrations were about three times as high as those of negative control mice. In TGM(rAOGEN)123 the transgene was highly expressed in liver and brain. Transcripts were also detected in heart, kidney and testis. In TGM(rAOGEN)92 the brain was the main expressing organ. In situ hybridization revealed an mRNA distribution in the brain of TGM(rAOGEN)123 similar to the one in rat. In TGM(rAOGEN)92 the expression pattern in the brain was aberrant. These data indicate that overexpression of the angiotensinogen gene in liver and brain leads to the development of hypertension in transgenic mice. The TGM(rAOGEN)123 constitutes a high angiotensin II type of hypertension and may provide a new experimental animal model to study the kinetics and function of the renin angiotensin system.
Candidiasis visualised by proteinase-directed immunofluorescence.
The secretory aspartic proteinases of Candida albicans and C. tropicalis are potential factors for virulence produced during infection. By indirect immunofluorescence, we have demonstrated proteinase antigen on elements of both species in deparaffinized tissue sections derived from clinical cases of mucosal and deep-seated candidiasis. Occasionally, we observed a halo of fluorescence in the close vicinity of candidal cells, which may reflect secretion of the enzyme. In kidneys, a ring of amorphous fluorescent material surrounding candidal colonies may illustrate alkaline denaturation of secreted enzyme within a pH gradient, which is generated by the fungus. Our findings support the view that candidal proteinase may be a diagnostically relevant antigen.
Markers for HIV-disease progression in untreated patients and patients receiving AZT: evaluation of viral activity, AZT resistance, serum cholesterol, beta 2-microglobulin, CD4+ cell counts, and HIV antigen.
In order to find parameters which allow the assessment of the clinical state of HIV patients with or without antiviral therapy, viral cultures on lymphocytes and monocytes/macrophages, CD4-cell counts, HIV antigen, beta 2-microglobulin and serum cholesterol were evaluated for their predictive value. As had been shown previously for lymphocytes, the efficiency of viral isolation on macrophages also depends on the disease stage (CDC) of the patients and thus has a high predictive value. A multivariant discriminant analysis showed that the combination of beta 2-microglobulin, viral antigen, CD4+ cell count and HDL cholesterol predicted the outcome of viral cultures with 80% accuracy. While viral antigen, CD4+ cell counts and beta 2-microglobulin had been known, HDL cholesterol deserves further evaluation as prognostic parameter. The analysis of HIV derived from patients with AZT showed a 20-200-fold in vitro drug resistance after seven to 24 months of therapy. DNA sequence determination of such strains isolated from AZT patients over time showed only two of the amino acid exchanges described in the literature for resistant strains and an additional Val60-Ile transition after 32 months of therapy.
Transgenic rats: new animal models in hypertension research. Invited lecture.
Explore the source record for details and available documents.
[Freud's interpretation of the manifest dream. Attempt at theoretical historical and biographical motives of ambivalent dream devaluation].
Freud's description of the manifest dream is characterized by an implicit appreciation and an explicit devaluation of dream images. The theoretical motives for this ambivalent evaluation are, according to the author, already apparent in Freud's primacy of the written word evident in his preanalytical writings, which submit dream images to the standard of the written word and therefore devaluate them. According to the author the underlying biographic motive is an ambivalent internalization of the jewish law that prohibits the creation of idols which was conveyed to Freud by his father and the Philippson Bible.
Biochemical and genetical analysis of AZT-resistant HIV-mutants.
Sequential virus isolates from an HIV-1-infected woman treated orally with 3'-azido-3'-deoxythymidine (AZT) for over two years showed a 10-fold reduced sensitivity for AZT after 8 months and a 100-fold resistance after 24-32 months of drug therapy. These AZT-resistant mutants were totally sensitive in vitro to other reverse transcriptase (RT)-inhibitors like the AZT-analogue 3'-fluoro-3'-deoxythymidine (FdT) or the chemically less related nucleoside analogue 2',3'-dideoxycytosine (ddC). Even the benzodiazepin derivative 4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)-imidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), a new drug specific for HIV-1 RT, was inhibitory for these virus strains. Moreover, compounds with different modes of action, e.g. polysulfated polyxylan, exhibited full antiviral activity as well. Thus, AZT resistance seems to be highly specific and should allow to develop further drugs to be used when AZT resistance has emerged. 5.9 kb fragments of the 5'-genomic halves of these sequential HIV-isolates were amplified by PCR and cloned. DNA sequence analysis revealed that the RT gene of the two highly AZT-resistant isolates carried two of the mutations described by Larder et al. [Science 246, (1989)], the Lys 70----Arg and the Thr 215----Tyr transitions. The isolate obtained after 32 months of AZT-therapy in addition contained a third mutation at position 67 (Asp----Asn); in contrast to Larder's report, no mutation was found at position 219. Thus, although these virus isolates showed at least a 100-fold reduced susceptibility for AZT in vitro, the four mutations postulated to be relevant for highly resistant strains were only partially confirmed.
Sexual dimorphism of blood pressure in spontaneously hypertensive rats: effects of anti-androgen treatment.
The mechanisms resulting in the greater predisposition of male subjects towards hypertension were investigated in different strains of rats with genetic hypertension [spontaneously hypertensive rats of the stroke-prone strain (SHRSP) and spontaneously hypertensive rats (SHR)] and their respective normotensive controls. Blood pressure was reduced in young (9 weeks of age) hypertensive rats by (1) surgical castration, (2) treatment with the testosterone receptor antagonist cyproterone acetate (CPA), which does not elevate testosterone, or (3) with the testosterone receptor antagonist flutamide, which leads to a feedback elevation of gonadotrophic hormones and plasma testosterone. These treatments had no effect on high blood pressure in old hypertensive rats aged 25 weeks. Both androgen receptor antagonists attenuated high blood pressure development when given for the first 10 days after birth. These data clearly relate the sexual dimorphism of hypertension to testosterone produced during male brain maturation in the early phase of hypertension development. Testosterone appears not to contribute directly to the maintenance of high blood pressure in established hypertension.