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Biomedical subjects

J Brunner

Publications and source records attributed to J Brunner.

At least 91 records · Page 5Linked to original sources
PubMed1991

The plasma membrane Ca2+ pump contains a site that interacts with its calmodulin-binding domain.

A synthetic, 28-residue peptide derived from the calmodulin-binding sequence of the plasma membrane Ca2+ pump (C28W) inhibits the ATPase activity of a calpain-produced, truncated fragment of the enzyme. The fragment, which has lost the calmodulin-binding domain, has a molecular mass of 124 kDa and is fully active in the absence of calmodulin. Replacement of Trp-8 in the peptide by an Ala decreases the overall inhibitory activity, while replacement with a Tyr increases it. However, at very low peptide concentrations the effect of Tyr replacement disappears. The synthetic peptide has been made photoactivatable by replacing Phe in position 9 with a synthetic phenylalanine analogue containing a diazirine group and was radioactively labeled by coupling a [3H]acetyl function to its N terminus. After cross-linking with the derivatized peptide, the 124-kDa fragment has been proteolyzed with either Lys-C, Asp-N, or V8 proteases, and the fragment(s) have been separated. Partial sequencing of the cross-linked, radioactive peptides has identified a site of the pump located C terminally to the phosphoenzyme-forming aspartic acid, spanning residues 537-544 of the hPMCA4 isoform of the enzyme. It is concluded that this sequence is part of a site which binds the calmodulin-binding domain of the pump.

Amino Acid Sequence↗
PubMed1991

Psychiatry, psychoanalysis, and politics during the First World War.

This article compares the therapeutic power used by psychiatrists and psychoanalysts serving in the German and Austro-Hungarian armies during World War I, and the ways in which their therapeutic techniques were related to governmental and military authority. When treating "shell shocked" soldiers, army psychiatrists were guided by nationalist commitment rather than concern for their patients. Whatever theoretical approach they took, they defined their therapeutic task as administrative intervention aimed to increase the docility of soldiers to the state and its military purposes. But violent means of coercion, designed to force neurotic soldiers back into duty, remained inefficient. Drawing from an alternative body of knowledge, army physicians influenced by Freud's writings, or trained in psychoanalysis, applied cathartic methods to treat the Central Powers' soldiers. Their approach was not only more humane, but also proved to be therapeutically more efficient.

Combat Disorders↗
PubMed1991

Only pharmacological doses of atrial natriuretic peptide affect intestinal ion transport in non-volume expanded rats.

Pharmacological doses of atrial natriuretic peptide were infused into rats to study its effect on intestinal transport. Saline control or two concentrations of rat alpha atrial natriuretic peptide (0.06 or 1.0 nmol/min/kg) were administered intravenously (1 ml) over one hour. Jejunal net transport of water and electrolytes was measured with a plasma-like luminal electrolyte solution using a 'closed loop' technique. Distal colonic potential difference and arterial blood pressure were monitored continuously. Blood samples for analysis of plasma atrial natriuretic peptide concentrations were taken at the end of the experiments. Plasma concentrations were increased (mean (SD) (2.1 (0.5) and 24.0 (1.1) nmol/l respectively) compared with the controls (0.023 (0.016) nmol/l). Blood pressure dropped by 30% (p less than 0.05) in both groups of rats receiving atrial natriuretic peptide but remained unchanged when control saline was infused. Jejunal net absorption was reduced (p less than 0.01) only in animals receiving the higher concentration of peptide (H2O from 173 (33) to 64 (69) microliters.h-1.cm-2, Na from 25.7 (5.3) to 10.9 (8.9) mumol.h-1.cm-2). Distal colonic potential difference was not affected by atrial natriuretic peptide. In conclusion massive doses of atrial natriuretic peptide are required to produce any change in intestinal salt and water transport in normal, non-volume expanded rats; these effects could be a non-specific or 'toxic' response.

Animals↗
PubMed1991

Atrial natriuretic peptide and water and electrolyte transport in the human jejunum.

The effects of atrial natriuretic peptide were investigated on water and electrolyte transport in the human jejunum. Six healthy male volunteers (aged 21-33 years) were studied using a triple lumen perfusion technique. A plasma like electrolyte solution containing polyethylene glycol (5 milligrams) as a non-absorbable marker was perfused into the jejunum at 10 ml/min, and net water and electrolyte transport and transepithelial potential difference were measured. Subjects were studied single blind on two occasions with either intravenous atrial natriuretic peptide (6 pmol/min/kg for 90 minutes) or placebo (saline), both after controlled sodium intake over three days. Plasma atrial natriuretic peptide concentrations rose from (mean (SD)) 10.3 (3.6) pmol/l to a peak of 96.0 (61.8) pmol/l. Jejunal net water and electrolyte fluxes and potential difference were identical in both the atrial natriuretic peptide and the control studies. Compared with placebo atrial natriuretic peptide induced a significantly greater diuresis (peak 10.2 (6.0) v 1.8 (1.0) ml/min, p less than 0.05) and natriuresis (peak 1069 (351) v 376 (208) mumol/min, p less than 0.01) and haemoconcentration (haematocrit 0.405 (0.040) v 0.368 (0.018), p less than 0.01). There was no difference in blood pressure, pulse rate, plasma electrolytes, and plasma osmolality between the two studies. There was no evidence to suggest an effect of atrial natriuretic peptide on jejunal water and electrolyte transport in healthy human subjects.

Adult↗
PubMed1991

Postmenopausal extragenital choriocarcinoma. A case report and review of the literature.

A case is presented of extragenital abdominal choriocarcinoma without uterine lesion in a postmenopausal woman. Nineteen years after her antecedent pregnancy, a legal abortion, and thirteen years after her menopause, the patient was admitted to the hospital because of intermittent abdominal pain. Explorative laparotomy revealed a large tumour mass in the greater omentum, mesosigmoideum, peritoneal implants and metastatic growth to the serosal lining of the uterus and the wall of the stomach. Cytoreductive surgery was performed. The histopathological report showed an extrauterine, nongonadal pure choriocarcinoma. Immunoperoxidase stain was strongly positive for hCG and a raised serum beta-hCG level preoperatively confirmed the diagnosis. A polychemotherapy regimen was administered. However, after six months the beta-hCG levels increased rapidly. Liver, lung and mediastinal metastases were diagnosed. The patient's condition rapidly deteriorated and she expired one month later. The post mortem examination showed a far advanced extragonadal pure choriocarcinoma without any obvious primary origin. The implications for a possible origin of extragonadal nongestational choriocarcinoma are briefly discussed.

Abdominal Neoplasms↗
PubMed1990

Isolation of isoproteins from monoclonal antibodies and recombinant proteins by chromatofocusing.

A fast protein liquid chromatographic method for the preparative separation of the various isoproteins is described. Highly purified human monoclonal antibodies, recombinant human superoxide dismutase and human superoxide dismutase from erythrocytes were used as starting material. The isoproteins were separated by chromatofocusing on Mono P columns. A very narrow pH gradient was applied to achieve complete separation of the isoproteins. The prepurification steps and the pretreatment of the samples to achieve optimum resolution are described in detail. The method is also applicable to extremely basic monoclonal antibodies (pI = 9). The successful separation was checked by isoelectric focusing in immobilized pH gradients (Immobilines). The future of these methods is discussed, because for many different biochemical and biophysical investigations pure and homogeneous isoproteins are necessary.

Antibodies, Monoclonal↗
PubMed1990

Interaction in situ of the cytoskeletal protein vinculin with bilayers studied by introducing a photoactivatable fatty acid into living chicken embryo fibroblasts.

The cytoskeletal protein vinculin, a putative actin--plasma-membrane linker, has been shown by hydrophobic photo-labeling to interact in vitro directly with bilayers of acidic phospholipids [Niggli et al. (1986) J. Biol. Chem. 261, 6912-6918]. In order to demonstrate that such an interaction occurs also in intact cells, chicken embryo fibroblasts were incubated for 2 h with a 3H-labeled photoactivatable fatty acid, 11-(4-[3-(trifluoromethyl)-diazirinyl]phenyl)-[2-3H]undecanoic acid. This resulted in biosynthetic incorporation into cellular lipids of a fraction of the fatty acid added. Following photolysis, vinculin was immunoprecipitated from different subcellular fractions using a specific polyclonal anti-vinculin antibody. The protein was recovered from both the cytosolic and the crude membrane fraction. Vinculin from both fractions incorporated label, but the membrane-associated population was at least eight times more strongly photolabeled than the cytosolic protein. Moreover, photolysis increased only labeling of the membrane-bound but not of the cytosolic protein. These results suggest that the direct interaction of vinculin with the hydrophobic core of the phospholipid layer observed in vitro may also be relevant in intact cells, and may be involved in its function as a linker protein.

Animals↗
PubMed1990

Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and perforin.

Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.

Affinity Labels↗
PubMed1989

Testing topological models for the membrane penetration of the fusion peptide of influenza virus hemagglutinin.

Low pH-induced binding of the bromelain-solubilized form of influenza virus hemagglutinin (BHA) to membranes occurs through the fusion peptide. From asymmetric hydrophobic photolabeling of membranes, evidence was obtained that this peptide penetrates only one leaflet of the bilayer. The asymmetrical labeling was achieved by employing a photoreactive analogue of a fatty acid whose transbilayer distribution can be manipulated by a membrane proton gradient.

Bromelains↗
PubMed1989

A 42K outer-membrane protein is a component of the yeast mitochondrial protein import site.

An engineered precursor protein that sticks in the import site of isolated yeast mitochondria can be specifically photo-crosslinked to a mitochondrial outer-membrane protein of relative molecular mass 42,000 (42K). This protein (termed import-site protein 42 or ISP 42) is exposed on the mitochondrial surface; antibodies against it block protein import into mitochondria. ISP 42 is the first identified component of the putative transmembrane machinery that imports proteins into mitochondria.

Biological Transport↗
PubMed1989

Hydrophobic binding of the ectodomain of influenza hemagglutinin to membranes occurs through the "fusion peptide".

Toward elucidating molecular details of virus-induced membrane fusion, we have studied the low pH-triggered interaction of the bromelain-solubilized ectodomain of influenza hemagglutinin with liposomes. Polypeptide segments which insert into the apolar phase of the lipid bilayer were first labeled specifically using either of the two membrane-restricted carbene-generating reagents, 3-(trifluoromethyl)-3-([125I]iodophenyl)diazirine and 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] undecanoyl]-sn-glycero-3-phosphorylcholine, and were then identified on the basis of cyanogen bromide and 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine-skatole fragment analysis and Edman degradations. Here, we demonstrate that the hydrophobic interaction is mediated solely by the so-called "fusion peptide" which corresponds to the NH2-terminal segment of the BHA2 subunit of nature influenza hemagglutinin. Predominant sites of labeling within that segment were Phe-3, Ile-6, Phe-9, Trp-14, Met-17, and Trp-21. The average 3-4 residue spacing between consecutive labeled amino acid side chains suggests a helical structure of that segment with an amphiphilic character.

Bromelains↗
PubMed1989

Need for plasma angiotensin measurements to investigate converting-enzyme inhibition in humans.

Since only a minute proportion of total angiotensin-converting enzyme (ACE) is present in plasma, the reliability of conventional in vitro measurements of ACE activity has been questioned. Data presented here demonstrate that the definition of ACE inhibition depends on the methodology used, with different results obtained with different substrates. We have developed a method that provides accurate and precise determinations of "true" angiotensin levels and in vivo ACE activity was estimated by measuring the plasma angiotensin II/angiotensin I ratio. Since the initial interruption of angiotensin II production by an ACE inhibitor stimulates renal renin release, the response can be quantitated by measuring changes in plasma levels of angiotensin I. The actual state of the renin-angiotensin system during ACE inhibition is represented by the plasma angiotensin II level. When ACE inhibition is no longer complete, increased angiotensin I levels bring the system back toward initial angiotensin II concentrations.

Administration, Oral↗
PubMed1989

Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers.

Synapsin I, a major neuron-specific phosphoprotein, is localized on the cytoplasmic surface of small synaptic vesicles to which it binds with high affinity. It contains a collagenase-resistant head domain and a collagenase-sensitive elongated tail domain. In the present study, the interaction between synapsin I and phospholipid vesicles has been characterized, and the protein domains involved in these interactions have been identified. When lipid vesicles were prepared from cholesterol and phospholipids using a lipid composition similar to that found in native synaptic vesicle membranes (40% phosphatidylcholine, 32% phosphatidylethanolamine, 12% phosphatidylserine, 5% phosphatidylinositol, 10% cholesterol, wt/wt), synapsin I bound with a dissociation constant of 14 nM and a maximal binding capacity of about 160 fmol of synapsin I/microgram of phospholipid. Increasing the ionic strength decreased the affinity without greatly affecting the maximal amount of synapsin I bound. When vesicles containing cholesterol and either phosphatidylcholine or phosphatidylcholine/phosphatidylethanolamine were tested, no significant binding was detected under any conditions examined. On the other hand, phosphatidylcholine vesicles containing either phosphatidylserine or phosphatidylinositol strongly interacted with synapsin I. The amount of synapsin I maximally bound was directly proportional to the percentage of acidic phospholipids present in the lipid bilayer, whereas the Kd value was not affected by varying the phospholipid composition. A study of synapsin I fragments obtained by cysteine-specific cleavage showed that the collagenase-resistant head domain actively bound to phospholipid vesicles; in contrast, the collagenase-sensitive tail domain, though strongly basic, did not significantly interact. Photolabeling of synapsin I was performed with the phosphatidylcholine analogue 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphocholine; this compound generates a highly reactive carbene that selectively interacts with membrane-embedded domains of membrane proteins. Synapsin I was significantly labeled upon photolysis when incubated with lipid vesicles containing acidic phospholipids and trace amounts of the photoactivatable phospholipid. Proteolytic cleavage of photolabeled synapsin I localized the label to the head domain of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Mischarging Escherichia coli tRNAPhe with L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine.

The Boc-protected derivative of a photoactivatable, carbene-generating analogue of phenylalanine, L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine [(Tmd)Phe], was used to acylate 5'-O-phosphorylcytidylyl(3'-5')adenosine (pCpA). A diacyl species was isolated which upon successive treatments with trifluoroacetic acid and 0.01 M HCl yielded a 1:1 mixture of 2'(3')-O-(Tmd)phenylalanyl-pCpA and of its 2'-5'-phosphodiester isomeric form. Adapting a procedure introduced by Hecht's group [Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., & Hecht, S.M. (1984) Biochemistry 23, 1468-1473], brief incubation of a 15 molar excess of this material with Escherichia coli tRNAPhe, missing at the acceptor stem the last two nucleotides (pCpA), in the presence of T4 RNA ligase and ATP afforded "chemically misaminoacylated" tRNAPhe in approximately 50% yield. Following chromatographic purification on DEAE-Sephadex A-25, benzoylated DEAE-cellulose, and Bio-Gel P-6, the misaminoacylated tRNAPhe was characterized by (i) urea-polyacrylamide gel electrophoresis, (ii) enzymatic reaminoacylation under homologous conditions following chemical deacylation, and (iii) its ability to stimulate protein synthesis in an in vitro translation system which, through the addition of the phenylalanyl-tRNA synthetase inhibitor phenylalaninyl-AMP, was unable to charge its endogenous tRNAPhe. The data demonstrate that we have prepared a biologically active misaminoacylated tRNAPhe.

Azirines↗
PubMed1988

Hydrophobic photolabeling identifies BHA2 as the subunit mediating the interaction of bromelain-solubilized influenza virus hemagglutinin with liposomes at low pH.

To investigate the molecular basis of the low-pH-mediated interaction of the bromelain-solubilized ectodomain of influenza virus hemagglutinin (BHA) with membranes, we have photolabeled BHA in the presence of liposomes with the two carbene-generating, membrane-directed reagents 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) and a new analogue of a phospholipid, 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl][2-3H] undecanoyl]-sn-glycero-3-phosphocholine ([3H]-PTPC/11). With the latter reagent, BHA was labeled in a strictly pH-dependent manner, i.e., at pH 5 only, whereas with [125I]TID, labeling was seen also at pH 7. In all experiments, the label was selectively incorporated into the BHA2 polypeptide, demonstrating that the interaction of BHA with membranes is mediated through this subunit, possibly via its hydrophobic N-terminal segment. Similar experiments with a number of other water-soluble proteins (ovalbumin, carbonic anhydrase, alpha-lactalbumin, trypsin, and soybean trypsin inhibitor) indicate that the ability to interact with liposomes at low pH is not a property specific for BHA but is observed with other, perhaps most, proteins.

Azirines↗
PubMed1987

Tetrameric detergent-soluble acetylcholinesterase from human caudate nucleus: subunit composition and number of active sites.

Purified tetrameric detergent-soluble acetylcholinesterase (DS-AChE) from human caudate nucleus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence as well as in presence of a reducing agent. Staining for protein revealed a main band at 66,000 daltons (light monomer) with additional bands at 78,000 daltons (heavy monomer) as well as 130,000 and 150,000 daltons (light and heavy dimers). The same four polypeptides were also detected by Western blotting and by autoradiography of [3H]diisopropylphosphoryl enzyme. Labeling of the enzyme with 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine showed that the heavy monomer contained the hydrophobic anchor of the enzyme, whereas the light monomer was practically not labeled. The hydrophobic anchor was susceptible to proteolytic degradation by proteinase K. The functional molarity of DS-AChE was determined by two independent methods. Four active sites for the tetrameric enzyme were estimated. The turnover number per site was 1.7 X 10(7) mol of acetylthiocholine iodide hydrolyzed X h-1.

Acetylcholinesterase↗