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D Neumann

Publications and source records attributed to D Neumann.

At least 91 records · Page 5Linked to original sources
PubMed1986

Mapping of the cAMP-dependent phosphorylation sites on the acetylcholine receptor.

We have synthesized a tetradecapeptide corresponding to residues 354-367 of the delta-subunit of Torpedo acetylcholine receptor. This peptide contains the sequence Arg-Arg-Ser-Ser which has been proposed as the site for phosphorylation of the acetylcholine receptor (AChR) by an endogenous cAMP-dependent protein kinase. We have shown that the synthetic peptide can be phosphorylated by the catalytic subunit of bovine heart cAMP-dependent protein kinase. Antibodies elicited against peptide 354-367 were shown to cross-react with native AChR and to bind specifically to the delta- and gamma-subunit as detected by immunoblotting. Furthermore, antipeptide antibodies were shown to inhibit specifically the cAMP-dependent phosphorylation of both the delta- and gamma-subunits. This suggests that the phosphorylation sites in the delta- and gamma-subunits are highly cross-reactive, and is in agreement with the demonstration that an endogenous cAMP-dependent kinase phosphorylates these two subunits, probably on homologous sequences. Tryptic digestion of the delta-subunit isolated from phosphorylated AChR yields a single 25-kd phosphorylated fragment. Immunoblotting experiments allowed us to map peptide 354-367 within this phosphorylated fragment.

Amino Acid Sequence↗
PubMed1986

Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor alpha subunit.

A synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo acetylcholine receptor alpha subunit, which contains the adjacent cysteine residues at positions 192 and 193, was recently shown by us to contain the essential elements for alpha-bungarotoxin binding. In the present study, we have used Sepharose-linked peptides for quantitative analysis of the cholinergic binding properties of this and other synthetic peptides. Sepharose-linked peptides corresponding to residues 1-20, 126-143, 143-158, 169-181, 185-196, 193-210, and 394-409 of the alpha subunit of Torpedo acetylcholine receptor, as well as a peptide corresponding to residues 185-196 of the alpha subunit of human acetylcholine receptor, were tested for their toxin-binding capacity. Of these immobilized peptides, only peptide 185-196 of the Torpedo acetylcholine receptor bound toxin significantly, thus verifying that this synthetic peptide contains essential components of the receptor toxin-binding site. Analysis of toxin binding to the peptide yielded a dissociation constant of 3.5 X 10(-5) M. This binding was inhibited by various cholinergic ligands. The inhibition potency obtained was alpha-bungarotoxin greater than Naja naja siamensis toxin greater than d-tubocurarine greater than decamethonium greater than acetylcholine greater than carbamoylcholine. This pharmacological profile resembles that of the nicotinic acetylcholine receptor and therefore suggests that the synthetic dodecapeptide also includes the neurotransmitter binding site. Reduction and carboxymethylation of the cysteine residues on peptide 185-196 inhibit its capacity to bind toxin, demonstrating that an intact disulfide is required for toxin binding. A decrease in toxin binding was also obtained following chemical modification of the tryptophan residue at position 187, thus implying its possible involvement in toxin binding. The failure to detect binding of toxin to the corresponding human sequence 185-196, in which the tryptophan residue is replaced by serine, supports this hypothesis.

Binding Sites↗
PubMed1986

Mapping of the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor.

Synthetic peptides and their respective antibodies have been used in order to map the alpha-bungarotoxin binding site within the alpha subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the alpha subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kDa toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any of the cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-Cys-Cys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the alpha subunit) was shown to bind alpha-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.

Amino Acid Sequence↗
PubMed1986

Coronary artery cardiomyopathy. Hemodynamic and prognostic implications.

To assess the prevalence and significance of left ventricular dilatation in patients with severe left ventricular dysfunction secondary to coronary artery disease (or coronary artery cardiomyopathy), we studied 70 patients with an ejection fraction of 35 percent or less and one-vessel coronary artery disease (n = 14) or with multivessel coronary artery disease (n = 56). None had had a recent myocardial infarction or valvular heart disease. Patients who underwent myocardial revascularization during follow-up were excluded. The left ventricular end-diastolic volume (measured by contrast ventriculography) was less than 110 ml/sq m in 14 patients (20 percent) (group 1), and was 110 ml/sq m or more in 56 patients (80 percent) (group 2). There were no differences between the two groups in age, sex, diabetes mellitus, hypertension, extent of coronary artery disease, or left ventricular asynergy. Patients in group 1 had lower pulmonary arterial wedge pressure (13 +/- 6 vs 22 +/- 10 mm Hg; p = 0.0008), lower left ventricular end-diastolic pressure (21 +/- 6 vs 27 +/- 9 mm Hg; p = 0.007), and higher left ventricular ejection fraction (31 +/- 2 vs 25 +/- 7 percent; p = 0.001) than patients in group 2. At a mean follow-up of 27 months, 24 patients had died of cardiac causes, all of whom were in group 2. Survival was significantly better in group 1 than in group 2 (Mantel-Cox, p = 0.009). Survival analysis (Cox models) of 20 clinical, hemodynamic, and angiographic variables showed that ejection fraction (chi2 = 13.6; p less than 0.001) and end-diastolic volume chi2 = 4.7; p = 0.03) were the most significant predictors of death. Thus, minimally dilated coronary artery cardiomyopathy is a distinct entity with favorable hemodynamics. Prognostically, the end-diastolic volume adds significant predictive information to the ejection fraction among conservatively treated patients.

Adult↗
PubMed1986

[Combined therapy with high-dosage cisplatin and radiation in advanced ENT tumors (a phase-II study)].

The most promising method to obtain at least a palliative effect in advanced ORL tumors seems to be a combination of chemotherapy and radiotherapy. We made an attempt to reach a higher remission rate (n = 28) by means of an extraordinary high dose of cis-platinum and the usual maximum radiation dose of 60 Gy: The method had to be abandoned because the rates of success (CR and PR) decreased from 79% to 46% in eight months, the side effects, however, were beyond a tolerable degree. We are of the opinion that the same results can be obtained by less radical methods, too.

Adult↗
PubMed1986

[Excentric rotation irradiation in ORL tumors].

A risk of dose insufficiency in the regions situated laterally to the spine and diagonally behind the spine is shown for the irradiation schemes applied hitherto in ORL tumors. In every case the fields are arranged in such a way that the myelon is not exposed to radiation. The excentric rotation technique presented guarantees that the exposition of the myelon doses not exceed 50% of the maximum dose. The axis lays within the myelon itself, and the central rays show an excentric deviation of 3 to 4 degrees. Thus a homogeneous dose distribution is achieved for the complete head and neck section.

Carcinoma, Squamous Cell↗
PubMed1985

[1 year's experience with a 57-cm image intensifier in a universal roentgen work station].

3342 patients were examined by means of a 57 cm image intensifier during one year. The installation permits examination with the patient standing or lying. The resolution was measured with a lead grid under conditions resembling actual practice and were found to be similar to those obtained with angiographic equipment. Using a medium format technique for examining the gut and vascular system resulted in radiographic quality which was not significantly different from the use of a large format technique. For examination of the skeleton, radiographic quality, using the intensifier, was markedly poorer than that resulting from Bucky examinations. There was no diagnostic difference when carrying out lymphograms. The large intensifier window of 53 cm was particularly advantageous for DSA of the pelvic and lower limb vessels when compared with smaller intensifiers. Possible savings of film and contrast medium costs are discussed.

Adult↗
PubMed1985

Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.

Synthetic peptides and their respective antibodies were used in an attempt to localize and identify the ligand-binding site of the nicotinic acetylcholine receptor. Two peptides of the receptor alpha subunit were synthesized, the first corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the first two cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies thus obtained cross-reacted with the receptor and bound specifically to its alpha subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the alpha-bungarotoxin (alpha-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor alpha subunit generated several fragments. Antipeptide (1-20) and antipeptide (126-143) both bound a 26-kDa fragment, whereas only antipeptide (126-143) bound a 17-kDa fragment. None of these fragments were found to bind alpha-BTX. On the other hand, alpha-BTX bound to an 18-kDa fragment that did not react with either of the antipeptide antibodies. Moreover, the 26-kDa and 17-kDa fragments were also found to contain the endoglycosidase H-susceptible oligosaccharide chain. Our results indicate that the toxin-binding site lies beyond the first possible V8 protease cleavage site after residues 126-143: i.e., Asp-152. This location is in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand-binding site.

Acetylcholine↗
PubMed1984

Target dependent and independent stages in regeneration.

Goldfish retinae deprived of their target by tectal ablation showed characteristics of regeneration, such as sprouting activity in culture and changes in protein synthesis. These features resemble changes occurring in regenerating retinae of optic nerves which were subjected only to crush injury. Retinae of tectal ablated nerves maintained their ability to sprout in culture longer than retinae of crushed-injured optic nerves. This may indicate a possible absence of the machinery which regulates growth termination as reflected in this case, by the enduring growth activity in culture. Among the changes in protein synthesis which occurred in retinae following either tectal ablation or crush injury, the most pronounced were in tubulin and in some other polypeptides of the following molecular weights: 46-49, 65 and 74 kdalton. Results are discussed with respect to the possible role of the target in initiating regeneration upon disconnection of the nerve, and in terminating growth upon reconnection.

Animals↗
PubMed1984

Anti-acetylcholine receptor response achieved by immunization with a synthetic peptide from the receptor sequence.

A synthetic peptide corresponding to the first twenty amino acids of the N-terminal region from the alpha-subunit of the Torpedo acetylcholine receptor cross reacts with antibodies to the receptor. A conjugate of this peptide to bovine serum albumin elicits in rabbits an immune response towards the synthetic peptide as well as towards the acetylcholine receptor. Blotting experiments demonstrate that the antipeptide antibodies react exclusively with the alpha-subunit of the acetylcholine receptor. Antibodies against synthetic peptides from various regions of the receptor sequence may provide useful reagents for structural and developmental analysis of the acetylcholine receptor as well as for the regulation of experimental autoimmune myasthenia gravis.

Animals↗
PubMed1984

Heat shock induced changes of plant cell ultrastructure and autoradiographic localization of heat shock proteins.

Treating tomato cell cultures and leaves by a physiological heat shock (hs) at 35 to 39 degrees C results in a progressive disintegration of the nucleolus and the assembly of cytoplasmic hs granules. Other ultrastructural changes are not observed. The alterations of the nucleoli coincide with an immediate stop of the processing and with a strongly decreased synthesis of pre-rRNA. Both hs effects are reversed after shift-down to normal temperature conditions (25 degrees C). Assembly of cytoplasmic hs granules depends on the accumulation of the newly forming hs proteins and on supraoptimal temperatures. It is not observed in preinduced cultures synthesizing hs proteins at 25 degrees C. Autoradiographic studies reveal the preferential accumulation of hsp in the nucleoli and hs granules. Furthermore uridine labeling points to the presence of RNA in electron dense particles of both subcellular components. A survey on the state of hsp synthesis and structural binding as well as on the ultrastructural changes is given for 12 selected hs regimes.

Autoradiography↗
PubMed1984

[Supine thoracic images with a mobile roentgen unit: comparison between the soft-ray and hard-ray technics].

To compare the influence of kilovoltage on the quality of supine chest roentgenograms obtained with a mobile x-ray unit, we examined the films of 50 patients on whom supine chest radiographs had been taken incidentally within a period of 24 hours both with high and with low-kVp technique. For evaluation of the image quality, a total of 11 criteria were compared and quantitated by 5 observers using a simple score method. The same advantages that have been attributed to the high-kVp technique on upright films were found on the corresponding supine examinations. These films showed a more balanced penetration and greater sharpness than those taken with low-kVp. The wider latitude of the high-kVp technique simplifies the exposure, and fewer repeat takes will therefore be necessary. The diagnostic gain outweights the few disadvantages, which are mostly coupled to the increase in scattered radiation. This, as well as the availability of more powerful and efficient mobile generators, should promote routine application of high-kVp technique for supine chest examinations.

Humans↗
PubMed1984

Dictyosomes participate in the intracellular pathway of storage proteins in developing Vicia faba cotyledons.

The main storage globulins of Vicia faba seeds, vicilin and legumin, were localized in ultrathin sections of developing cotyledons employing the protein A/colloidal gold-technique. Double labelling experiments with monospecific immunoglobulins directed against vicilin or legumin using different size classes of protein A-colloidal gold showed that both globulins are associated with protein lumps in the vacuoles of early developmental stages of the seeds and with the rough endoplasmic reticulum, the site of storage protein biosynthesis. Furthermore, vicilin and legumin have been detected in protein-containing vesicles in the cytoplasm as well as in dictyosomes and the dictyosome-derived vesicles. The association of storage globulins with the dictyosomes was also indicated by cell fractionation and immunoblotting of organelle contents derived from microsome, enriched dictyosome and protein body preparations. Protein blotting analyses employing a Concanavalin A/peroxidase technique showed that certain vicilin polypeptides were already glycosylated in the rough endoplasmic reticulum. Another part of the vicilin fraction as well as legumin polypeptides did not show any affinity to Concanavalin A. These data provide a direct evidence for a transient involvement of dictyosomes in the intracellular pathway of legume storage globulins from the rough endoplasmic reticulum to their accumulation site in the protein bodies.

Cell Fractionation↗
PubMed1983

Regulation of mRNA levels for microtubule proteins during nerve regeneration.

The molecular regulation of tubulin synthesis was investigated in the regenerating goldfish retina. Previous in vivo studies pointed to an increase in tubulin synthesis in the retina during regeneration of the injured goldfish optic nerve. Using labeled cDNA probes, we showed that this increase occurs as a result of enhanced tubulin mRNA levels. Analysis of labeled in vivo products revealed enhanced beta 2-tubulin synthesis accompanied by an increase in the level of the low-Mr microtubule-associated proteins identified as TAU factors. The results are discussed with respect to the possible involvement of these proteins in the process of nerve regeneration.

Animals↗
PubMed1983

Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.

Cytoplasmic Granules↗
PubMed1982

Changes in cytoplasmic ultrastructure during submerged cultivation of a peptide alkaloids-producing strain of Claviceps purpurea (Fr.) Tul.

A strain of Claviceps purpurea, designated Pepty 695/S produces ergotoxine alkaloids under particular conditions of fermentation. The onset of alkaloid synthesis occurs around the second day of cultivation. Alkaloid formation is connected with morphological and ultrastructural changes. In the first 3-5 days of cultivation short thickened, septated hyphae, organized in plectenchymatic pellets as well as large single cells are formed. The hyphae are ultrastructurally characterized by increasing number of lipid droplets, deposits of glycogen and by extended ER membranes, which apparently may form numerous vesicles. The correlations between lipid and alkaloid synthesis are discussed.

Cell Nucleus↗