Biomedical subjects
Publications and source records attributed to G Müller.
The lethal white foal syndrome (congenital intestinal aganglionosis) was diagnosed by history, clinical signs and pathological findings in a female foal, born in March 1992, that was an offspring of two overo-spotted paint horses. The syndrome is a congenital innervation defect of the gastrointestinal tract. A literature review of this condition, relatively unknown in Germany, is given.
PURPOSE: Examination of the influence of total tumor dose, additional therapy and age on the development of radiation pneumonitis and lung fibrosis. PATIENTS AND METHODS: Two hundred and sixteen patients with squamous cell carcinoma of the lung were examined retrospectively. Hundred and eighty-three of them received a percutaneous radiation therapy of the primary tumor and the mediastinal and ipsilateral hilar lymph nodes. The majority of 130 patients was given radiotherapy as only treatment, 27 were irradiated after resection and 26 received chemo- and radiotherapy. Treatment volume was determined by three-dimensional planning using chest CT-scan. The irradiation was administered in a first series of 56 Gy and a following boost to the primary and involved lymph nodes to a total dose of 70 Gy. There was one daily irradiation with 2 Gy 5 days a week. Post-resectional radiation encompassed the bronchus stump and the mediastinum with a total dose of about 60 Gy. RESULTS: Sixty-eight of the 183 irradiated patients developed a pneumonitis (37.2%) and 60 a lung fibrosis (32.8%). The most important factor was the administered total irradiation dose (p < 0.001). There was no pneumonitis documented below 36 Gy and no fibrosis below 40 Gy. An additional chemotherapy did not increase the incidence (pneumonitis after radiotherapy 35.4%, after radio- and chemotherapy 34.6%; fibrosis after radiotherapy 33.9%, after radio- and chemotherapy 38.5%). Particularly high was the incidence of pneumonitis when radiotherapy followed resection (48.2%) while the rate of fibrosis in this group of patients was relatively low (22.2%). Age of patients did not affect the frequency of pneumonitis while fibrosis significantly occurred more often with increasing age (p = 0.037). CONCLUSION: The total dose affecting the normal lung tissue is the most important factor for the development of radiation pneumonitis and lung fibrosis. It is recommended to keep the treatment volume appropriate to the individual extend of tumor. Good results are achieved with a computer supported CT-planning which helps to gain minimal burdening of normal tissue.
Duodenal diverticula are found in up to 22 percent of the population. According to the literature, less than 10 percent of duodenal diverticula will cause symptoms, and about 1 percent will require definite treatment. There is no indication for treatment of asymptomatic patients with duodenal diverticula. A trial of conservative treatment is justified if the symptomatic patient is in stable condition. If symptoms persist or serious complications are present and other pathology of the abdomen that might be responsible is ruled out, definitive treatment is necessary. For intraluminal duodenal diverticula an elective endoscopic approach is possible. Our approach for extraluminal duodenal diverticula is discussed herein. The clinical presentations of 50 patients from 1986 to 1992 are summarized. In a surprisingly high proportion (12 percent), the symptoms could be clearly related to the duodenal diverticula. Eight percent were treated surgically, and postoperative mortality rates zero. In the literature, postoperative mortality rates even for elective surgery are 8 to 12 percent and reach 20 percent for emergency operations.
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The anatomical localization of the Na+/bile acid cotransport system from rabbit small intestine was determined using brush border membrane vesicles prepared from eight different segments of the small intestine. Na(+)-dependent transport activity for bile acids, both for [3H]taurocholate and [3H]cholate, was found in the distal segment 8 only representing the terminal 12% of the small intestine. In contrast, the Na(+)-dependent D-glucose transporter and the H(+)-dependent oligopeptide transporter were found over the whole length of rabbit small intestine in all segments. Photoaffinity labeling with 7,7-azo- and 3,3-azo-derivatives of taurocholate with subsequent fluorographic detection of labeled polypeptides after one- and two-dimensional gel electrophoresis showed that an integral membrane polypeptide of M(r) 87,000 is present in the entire small intestine, whereas an integral membrane protein of M(r) 93,000 together with a peripheral membrane protein of M(r) 14,000 are exclusively expressed in the distal small intestine correlating with Na(+)-dependent bile acid transport activity. Photoaffinity labeling with the cationic bile acid derivative 1-(7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta[3 beta-3H]cholan-24-oyl)-1,2- diaminoethane hydrochloride and 7,7-azo-3 alpha,12 beta-dihydroxy-5 beta[12 alpha-3H]cholan-24-oic acid did not result in a specific labeling of the above mentioned proteins, demonstrating their specificity for physiological bile acids. Photoaffinity labeling of the 93- and 14-kDa bile acid-binding proteins was strongly Na(+)-dependent. Significant labeling of the 93- and 14-kDa proteins occurred only in the presence of Na+ ions with maximal labeling above 100 mM [Na+] showing a parallel [Na+] dependence to transport activity. Inactivation of Na(+)-dependent [3H]taurocholate uptake by treatment of ileal brush border membrane vesicles with 4-nitrobenzo-2-oxa-1,3-diazol chloride led to a parallel decrease in the extent of photoaffinity labeling of both the 93- and 14-kDa protein. Sequence analysis of the membrane-bound 14-kDa bile acid-binding protein surprisingly revealed its identity with gastrotropin, a hydrophobic ligand-binding protein exclusively found in the cytosol from ileocytes and thought to be involved in the intracellular transport of bile acids from the brush border membrane to the basolateral pole of the ileocyte. In conclusion, the present studies suggest that both an integral 93- and a peripheral 14-kDa membrane protein, identified as gastrotropin, and both exclusively expressed in the terminal ileum, are essential components of the Na+/bile acid cotransport system in rabbit terminal ileum.
The efficacy of intravenous metoprolol in preventing symptoms during a repeat tilt test was compared with the outcome of chronic oral treatment in 21 patients (14 female, 7 male), age 8 to 20 years (mean 13 +/- 3) with unexplained syncope (> or = 1 episode) and a positive response to tilt testing. A positive response was defined as the development of either syncope or presyncope. During the initial tilt test, a positive response occurred during baseline (14 patients) or isoproterenol (0.03 to 0.1 microgram/kg/min) infusion (7 patients) with a cardioinhibitory (1 patient), vasodepressor (5 patients) or mixed (15 patients) pattern. Metoprolol (0.1 to 0.2 mg/kg) was administered intravenously. During the repeat tilt test, response was negative in 18 patients, including 11 of 14 patients with a positive response in the baseline and 7 of 7 patients with a positive response during isoproterenol infusion. Metoprolol (0.8 to 2.8 mg/kg/day) was administered orally to 15 patients for an average of 10 months. Symptoms were absent (7 patients) or improved (2 patients); metoprolol was discontinued because of adverse effects (3 patients) or recurrence of symptoms (3 patients). In 7 of 12 patients with a negative response and 2 of 3 patients with a positive response after intravenous metoprolol, oral administration of metoprolol prevented or improved symptoms without adverse effects. Many young patients (60%) with recurrent syncope obtained symptomatic improvement from chronic oral metoprolol treatment without adverse effects; repeat tilt testing after intravenous metoprolol did not appear to offer any additional information than would have been obtained from a trial of chronic oral treatment.
Chimeras of the double chain bis-cystinyl hinge fragment 225-232/225'-232' of the human IgG1 and of peptides related to human little-gastrin were synthesized, whereby the fully bioactive gastrin sequences 2-17 and 5-17 were amide-bond-linked N- and N- or C-terminally, respectively, to the hinge peptide. All the dimeric constructs proved to be efficient immunogens; however, both the configuration of the constructs and the length of the haptenic gastrin molecule were found to drastically affect the specificity of the antibody response and, thus, the type of dominant immune epitope expressed. The different degree of accessibility of the gastrin chains in the dimers is similarly reflected by their binding affinities to gastrin receptors and their bioactivities in vivo. Molecular dynamics simulations of the chimeric compounds clearly revealed that the conformational space of the gastrin peptide chains 2-17 and 5-17 is strongly restricted upon linkage to the hinge peptide. Only in the gastrin-(2-17) construct does sufficient free conformational space seem to be retained, at least for one of the two gastrin chains, in order to allow folding into the bioactive structure. This also agrees with the observation that the dimeric gastrin-(2-17) behaves like a gastrin monomer in terms of receptor binding affinity and biopotency in vivo; but it could additionally explain why an antibody response of gastrin receptor-like specificity could only be induced with this construct. The experimental data may therefore suggest a high degree of parallelism between the mechanism of recognition of the gastrin peptides in the dimeric constructs as hormonal ligands by the gastrin receptors and as haptens by the immune competent cells.
We have purified bleomycin hydrolase from yeast (molecular mass 55,000 Da). Using protein sequence-derived degenerate oligonucleotide primers and amplification by polymerase chain reaction, the yeast gene BLH1 was isolated and characterized. The deduced amino acid sequence (483 amino acids) exhibits surprisingly high homology to vertebrate bleomycin hydrolase (43% identical residues and 22% conserved exchanges). It contains three blocks of sequences found conserved in other members of the thiol proteinase family and thought to be associated with the catalytic centre. BLH1 is non-essential under all growth conditions tested. However, in the presence of 3.5 mg bleomycin/ml medium wild-type cells have a slight growth advantage compared to blh1 mutant cells.
Sulphonylurea drugs stimulate glucose transport and metabolism in muscle and fat cells in vitro. The molecular basis for the insulin-mimetic extrapancreatic effects of these oral antidiabetic therapeutic agents is unknown at present. Here we demonstrate that incubation of 3T3 adipocytes with the novel sulphonylurea, glimepiride, causes a time- and concentration-dependent release of the glycosylphosphatidylinositol (GPI)-anchored ecto-proteins, 5'-nucleotidase, lipoprotein lipase and a 62 kDa cyclic AMP (cAMP)-binding protein from the plasma membrane into the culture medium. The change in the localization is accompanied by conversion of the membrane-anchored amphiphilic proteins into their soluble hydrophilic versions, as judged by pulse-chase experiments and Triton X-114 partitioning, and by appearance of anti-cross-reacting determinant (CRD) immunoreactivity of the released proteins as shown by Western blotting. Metabolic labelling of cells with myo-[14C]inositol demonstrates that inositol is retained in the major portion of released lipoprotein lipase and cAMP-binding ectoprotein. The identification of inositol phosphate after deamination of these proteins with nitrous acid suggests cleavage of their GPI membrane anchor by a GPI-specific phospholipase C. However, after longer incubation with glimepiride the amount of soluble versions of the GPI-proteins lacking inositol and anti-CRD immunoreactivity increases, which may be caused by additional drug-stimulated hydrolytic events within their GPI structure or C-termini. Since insulin also stimulates membrane release of these GPI-modified proteins, and in combination with glimepiride in a synergistic manner, sulphonylurea drugs may exert their peripheral actions in adipose tissue by using (part of) the insulin postreceptor signalling cascade at the step of activation of a GPI-specific phospholipase C.
To investigate the role of proline in defining beta turn conformations within cyclic hexa- and pentapeptides we synthesized and determined the conformations of a series of L- and D-proline-containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L-proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D-proline peptides. The cis conformations of the compounds cyclo(-Pro-Ala-Ala-Pro-Ala-Ala), cyclo(-Arg-Gly-Asp-Phe-Pro-Gly-), cyclo(-Arg-Gly-Asp-Phe-Pro-Ala-), cyclo (-Pro-Ala-Ala-Ala-Ala-), and cyclo(-Pro-Ala-Pro-Ala-Ala-) form uncommon beta VI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline-following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg-Gly-Asp), are incorporated.
N7-Phenylguanine, a base adduct possibly formed after arylation of DNA by benzene oxide, the first reaction metabolite during benzene metabolism, was synthesized in our laboratory and used as reference for the production and characterization of monoclonal antibodies. 2-Hydroxymethyl-7-phenylhypoxanthine, a molecule structurally similar to N7-phenylguanine, was coupled by a linker molecule to different carrier proteins. The resulting conjugate was used to immunize BALB/c mice, the spleen cells of which were fused with mouse P3X63-Ag8.653 myeloma cells to obtain monoclonal antibodies. Several hybridoma lines were cultivated in defined media and characterized as to sensitivity and specificity by an enzyme-linked immunosorbent assay (ELISA). Competitive ELISA demonstrated that all antibodies showed a very high affinity for N7-phenylguanine but had a lower affinity towards various other samples including N7-chlorophenylguanines and C8-, N2-and O6-phenylguanine. As little as about 20 pg N7-phenylguanine could be detected with one of the most sensitive antibodies, CE6/G11, with a colorimetric end point while the detection limit could be lowered to about 10 pg N7-phenylguanine when a fluorescent end point was used. The detection limit of other methods used to determine N7-phenylguanine so far is 10 ng for gas-chromatography/mass-spectrometry and 1 ng for high-pressure liquid chromatography. Thus the use of specific monoclonal antibodies seems to be the most sensitive method for the detection of N7-phenylguanine.
Fifteen maxillary central incisors were treated in vitro with pulsed CO2 laser radiation (wavelength:9.6-microns pulse duration:135-microseconds pulse energy:60 mJ energy density:12 J/cm2) delivered by an AgCl fiber into the root canal. Preliminary results show opening of dentin tubules as well as fused areas of hydroxyapatite in the root canal after laser treatment. Temperature measurement at the root surface showed that 40 degrees C was not exceeded. These preliminary results show the ability of this laser system to open dentin tubules and to fuse hydroxyapatite but further development in fiber technology is necessary to achieve predictable results.
In the yeast Saccharomyces cerevisiae an amphiphilic cAMP-binding protein has been found recently to be anchored to plasma membranes by virtue of a glycolipid structure (Müller and Bandlow, 1991a, 1992). The cAMP-binding parameters of this protein are affected by the lipolytic removal of the glycosylphosphatidylinositol (GPI) membrane anchor by exogenous (G)PI-specific phospholipases C or D (PLC or PLD) (Müller and Bandlow, 1993) suggesting a regulatory role of glycolipidic membrane anchorage. Here we report that transfer of yeast cells from lactate to glucose medium results in the conversion of the amphiphilic form of the cAMP receptor protein into a hydrophilic version accompanied by the rapid loss of fatty acids from the GPI anchor of the [14C]palmitic acid-labeled protein. Analysis of the cleavage site identifies [14C]inositol phosphate as the major product after treatment of the soluble, [14C]inositol-labeled protein with nitrous acid which destroys the glucosamine constituent of the anchor. Together with the observed cross-reactivity of the hydrophilic fragment with antibodies directed against the cross-reacting determinant of soluble trypanosomal variable surface glycoproteins (i.e., myo-inositol-1,2-cyclic phosphate) this demonstrates that, in membrane release, the initial cleavage event is catalyzed by an intrinsic GPI-PLC activated upon transfer of cells to glucose medium. Release from the plasma membrane in soluble form requires, in addition, the presence of high salt or alpha-methyl mannopyranoside, or the removal of the carbohydrate moieties, because otherwise the protein remains associated with the membrane presumably at least in part via its N-glycosidic carbohydrate side chains. The data point to the possibility that cleavage of the anchor could play a role in the transfer of the signal for the nutritional situation to the interior of the cell.
Investigations with [14C]benzene indicate the formation of base adducts in vivo. Experiments to separate adducts from urine of [14C]benzene-exposed rats suggest the excretion of eight labeled compounds different from benzene metabolites. In order to obtain information about their structure we synthesized N7-, O6-, C8- and N2-phenylguanine. With regard to their chromatographic properties we compared these phenylguanines with products obtained by alkylation of guanine by metabolites of unlabeled and 14C-labeled benzene in vivo with HPLC with UV detection and liquid scintillation counting. Furthermore GC/MS and ELISA techniques were used to detect N7-phenylguanine. Phenylguanines could not be identified in collected DNA fractions. The labeled compounds detected in urine of [14C]benzene-exposed rats also showed deviations from the HPLC elution patterns of our reference substances. Even N7-phenylguanine, formerly suspected to be a urinary metabolite of benzene in the rat, could not be detected with these refined HPLC methods. With GC/MS a compound was found in trace amounts in concentrated rat urine samples, which had a similar fragmentation pattern to N7-phenylguanine. These data could not be confirmed by a sensitive immunological assay (ELISA). No N7-phenylguanine was detected in purified rat urine samples. The results suggest the excretion of a hydroxylated phenylguanine which may be formed in liver or bone marrow DNA by highly reactive hydroxylated intermediates. The OH group might be lost because of the high temperatures during GC/MS measurements. A hydroxy group at the phenyl-ring of N7-phenylguanine will cause other elution properties in HPLC compared to N7-phenylguanine.
We have developed a new genetic selection system for Tet repressor mutations with a noninducible phenotype for tetracycline (TetRs). Extensive chemical mutagenesis of tetR yielded 93 single-site Tet repressor mutations. They map from residue 23 preceding the alpha-helix-turn-alpha-helix operator binding motif to residue 196 close to the C terminus of the repressor. Thirty-three of the mutations are clustered between residues 95 and 117, and another 27 are clustered between residues 131 to 158. Several of the mutants were characterized quantitatively in vivo for induction by tetracycline and anhydrotetracycline. While all of these are severely reduced in tetracycline-mediated induction, only some of them are affected for anhydrotetracycline-mediated induction.
In a retrospective study a cohort of 2,277 patients was defined by colonoscopy. Among other factors it was tested whether in these patients laxative use or the endoscopically diagnosed presence of melanosis coli were risk factors related to colorectal neoplasma. In comparison to patients taking no laxatives there was no significant increase in colorectal cancer rate either in laxative users or in patients with melanosis coli. However, there was a statistically significant association between the occurrence of colorectal adenomas and laxative use (relative risk of all patients exposed to laxatives = 1.72; of patients exposed to laxatives without melanosis coli = 1.47). The relative risk of adenoma development in patients with melanosis coli was 2.19. Taking into account that polyps can be diagnosed in the dark mucosa of melanosis coli patients more easily, even this relative risk of 2.19 seems to be related to a generally enhanced risk of laxative intake rather than to a special group of (anthranoid containing) laxatives.
The differentiation-dependent expression of purinergic receptors for metabolically stable analogues of adenosine and ATP was studied in human mononuclear phagocytes (MNPs). Ligands of these receptors are able to modulate cellular cholesterol metabolism. In addition, the intracellular signal transduction pathways of the purinergic receptor system were examined. ATP gamma S, the metabolic stable analogue of ATP, was used as a P2 ligand, and 2-p-(2-carboxyethyl)phenylethylamino-5'-N-ethylcarboxamido adenosine (CGS 21680) and 5'-(N-ethylcarboxamido)adenosine (NECA) were used as P1 ligands in binding studies. Binding of [35S]ATP gamma S to MNPs at 4 degrees C revealed saturable low-affinity binding sites with a Kd of 868 +/- 52 nmol/L and Bmax of 7.3 +/- 0.4 pmol per 10(6) cells in 1-day cultured human MNPs and a Kd of 780 +/- 30 nmol/L and Bmax of 14.0 +/- 0.8 pmol per 10(6) cells in 7-day cultured human MNPs. The characterization of the P1 receptors on 1- and 7-day cultured human MNPs showed that they are expressed only on 7-day cultured human MNPs. The specific binding curve of the adenosine A2 receptor agonist [3H]CGS 21680 was biphasic, with a Kd1 of 33 +/- 15 nmol/L and a Kd2 of 90 +/- 10 nmol/L and with Bmax1 of 0.19 +/- 0.06 pmol per 10(6) cells and Bmax2 of 0.41 +/- 0.09 pmol per 10(6) cells, whereas NECA did not exhibit specific binding. The typical agonists for probing A1 receptor subtypes did not bind to 1- and 7-day cultured human MNPs, indicating that only A2 receptors are expressed on 7-day cultured human MNPs. ATP gamma S enhanced [Ca2+]i in 1- and 7-day cultured human MNPs in a concentration-dependent manner, whereas the P1 ligands, adenosine and CGS 21680, induced Ca2+ flux only in 7-day cultured MNPs. All three drugs increased intracellular cAMP levels in 7-day cultured human MNPs at a concentration of 10(-5) mol/L, whereas no effect was observed in 1-day cultured human MNPs. The uptake of fluorescently labeled acetylated low-density lipoprotein (LDL) in 7-day cultured human MNPs was inhibited by adenosine, CGS 21680, ATP, and ATP gamma S. No significant influence of these compounds was measured on the uptake of LDL, acetylated LDL, and high-density lipoprotein, in 1-day cultured MNPs. Our investigations indicate that the expression of P2y and A2 receptors is increased during differentiation of blood monocytes to macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)