Retraction. Functional and nonfunctional measles virus matrix genes from lethal human brain infections.
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Retraction Notice↗
Biomedical subjects
A Schmid
Publications and source records attributed to A Schmid.
Functional and nonfunctional measles virus matrix genes from lethal human brain infections.
Subacute sclerosing panencephalitis (SSPE) is a lethal disease induced by the persistence of measles virus in the human brain. In many SSPE cases, the viral matrix (M) protein cannot be detected; in others, M proteins of the expected size are found and sequence analysis of M cDNAs has confirmed that the reading frames are intact, showing only several missense mutations. To determine whether these alterations result in nonfunctional proteins, we have replaced the M gene of an infectious full-length genomic cDNA (from vaccine strain Edmonston) with different M genes derived from four patients with SSPE. One of the SSPE M genes tested proved to be functionally competent, giving rise to a virus yielding titers similar to those of viruses containing the M gene from control lytic strains. The other three SSPE M genes were not functionally competent in the same test. In all three cases, the inactivating changes resided in the carboxyl-terminal half of the M protein, as shown by the exchange of either of the two genes halves. In summary, mutational M gene alterations, which either prevent synthesis of M protein altogether or only allow synthesis of nonfunctional M protein, have been detected by us and by others in 9 of 10 SSPE cases. The one functional M gene appears to be an exception to the rule, indicating that M gene alteration might not be an absolute requirement for disease development.
Animals↗
Effects of varying physical parameters of a PET system on the accuracy of radioactivity quantitation in vivo.
The accuracy of quantitation of radioactivity by positron emission tomography (PET) is dependent on design parameters which differ among the PET-cameras in use today. Using a high resolution PET camera (Scanditronix PC 4096-15WB) we have simulated various of these parametric design differences such as xy- and z-resolution, slice thickness, methods of scatter and attenuation correction. The quantitation of actual patient data differed by up to 40% for both static and dynamic data when single parameters were varied to fit cameras of preexisting and recent design. For example, we found that cortical FDG data are 15% lower for 11 mm image resolution than for 5.5 mm image resolution, and striatal uptake of a D2-receptor-ligand decreases by 25% when the slice thickness is 14 mm instead of 7 mm. In general, the errors due to the single effects studied here are expected to be cumulative, leading to an even greater discrepancy in the overall performance of older PET cameras when compared to those of recent design.
Brain↗
Identification and characterization of angiotensin II receptor subtypes in rabbit ventricular myocardium.
CGP 42 112 A and DuP 753 block [125I]-angiotensin II binding in rabbit ventricular myocardial membranes in a clearly biphasic manner, indicating the existence of high- and low-affinity sites for these highly selective agents. Assays using concentrations of either agent large enough to prevent high-affinity binding show that their respective high-affinity sites are distinct, and each corresponds to the low-affinity site of the other. The two receptor subsets, present in nearly equal proportions, are also distinguishable by their different sensitivities to dithiothreitol. These findings afford strong evidence for the existence of two distinct angiotensin II receptors in rabbit myocardium, corresponding to the A and B subtypes recently described in adrenals.
Angiotensin II↗
Pore formation associated with the tail-tip protein pb2 of bacteriophage T5.
Upon binding of bacteriophage T5 tails to purified FhuA receptor protein the tail-tip protein pb2 became extremely sensitive to trypsin and other proteases. However, when T5 tails were bound to FhuA integrated into liposomes, pb2 was found to retain some resistance to trypsin. Electron microscopic examination of tail-liposome complexes supported the idea that trypsin resistance of pb2 in such complexes was caused by insertion of the tail-tip into the liposomes. pb2 was isolated from tails by treatment with sodium dodecyl sulfate and was further purified by gel filtration using a fast protein liquid chromatography system. pb2 obtained with this procedure was most likely monomeric. It was extremely sensitive to trypsin. When reconstituted into black lipid bilayer membranes, it formed pores with an average single-channel conductance of 4.6 nanosiemens in 1 M KCl. Zero-current potential measurements showed only a very slight preference, if any, for cations over anions. The data are compatible with pb2 forming a large water-filled transmembrane channel. The functioning during infection of pb2 in cytoplasmic membrane depolarization and phage DNA uptake into the cell is discussed.
Electric Conductivity↗
Voltage-dependent InsP3-insensitive calcium channels in membranes of pancreatic endoplasmic reticulum vesicles.
Stimulus-secretion coupling in exocrine glands involves Ca2+ release from intracellular stores. In endoplasmic reticulum vesicle preparations from rat exocrine pancreas, an inositol 1,4,5-trisphosphate(InsP3)-sensitive, as well as an InsP3-insensitive, Ca2+ pool has been characterized. But Ca2+ channels in the endoplasmic reticulum of rat exocrine pancreas have not been demonstrated at the level of single-channel current. We have now used the patch-clamp technique on endoplasmic reticulum vesicles fused by means of the dehydration-rehydration method. In excised patches, single Ba2(+)- and Ca2(+)-selective channels were recorded. The channel activity was markedly voltage-dependent. Caffeine increased channel open-state probability, whereas ruthenium red and Cd2+ blocked single-channel currents. Ryanodine, nifedipine and heparin had no effect on channel activity. The channel activity was not dependent on the free Ca2+ concentration, the presence of InsP3, or pH. We conclude that this calcium channel mediates Ca2+ release from an intracellular store through an InsP3-insensitive mechanism.
Animals↗
Hypertension and sports activities: institutional experience.
Findings of 185 patients and 271 control subjects are presented for the assessment of work capacity in hypertensive individuals (primary hypertension); an attempt at classification by hypertensive stage is seen as an essential presupposition. The subdivision into three stages recommended by the Experts' Commission of the WHO places the effects of hypertension on the organism, especially on the heart, in the focal point. This appears justified from a prognostic, pathophysiological, and therapeutic point of view. Hemodynamics in the examined patients undergo increasing impairment in relation to the stage of hypertension with a decrease in maximum cardiac index and work capacity and an increase in myocardial oxygen requirement. Initially, only the diastolic cardiac function is impaired; however, in advanced stages, the systolic function of the heart is impaired as well. Evaluation of work capacity is usually possible from a cardiac point of view by means of noninvasive echocardiographic and spiroergometric methods. The mass/volume ratio of the left cardiac ventricle and the relationship between left ventricular muscle mass (or volume) and work capacity are especially important. Both experience characteristic changes depending on the stage of hypertension and thus permit precise determination of work capacity, progress controls, and delineation from physiological cardiac hypertrophy (athletic heart).
Cardiomegaly↗
Infectious measles virus from cloned cDNA.
The study of measles virus (MV) and of negative strand RNA viruses in general has been hampered by the lack of an experimental system for genetic manipulation. Here we describe a procedure for generating infectious MV from cloned MV cDNA. First we assembled a genetically marked DNA copy of the MV genome in plasmids, under the control of phage T3 or T7 promoters, allowing production of transcripts almost identical to the MV genome or antigenome. Incubation of these linearized plasmid DNAs with the appropriate phage polymerase and only two ribonucleoside triphosphates yielded committed transcription complexes. Microinjection of these complexes into the cytoplasm of helper cells which provide the proteins necessary for MV genome encapsidation and transcription/replication, reproducibly give rise to lytic MVs. The transcripts of one of these viruses were analysed by sequencing after reverse transcription followed by DNA amplification, and found to contain the genetic tags. The described procedure permits the analysis of a negative strand RNA virus with the same genetic tools previously applicable only to positive strand RNA viruses and retroviruses.
Base Sequence↗
The Drosophila neurogenic locus mastermind encodes a nuclear protein unusually rich in amino acid homopolymers.
The neurogenic loci of Drosophila are required for proper partitioning of ectodermal cells into epidermal versus neural lineages. The loci appear to encode components of a developmental pathway involving cellular communication. In an effort to understand the role of the neurogenic locus mastermind in these processes, we have characterized its expression and sequence. The locus produces a number of transcripts that accumulate ubiquitously during early embryogenesis but more specifically in the central nervous system during later stages. Sequence analysis of a major cDNA product predicts an unusual protein containing an abundance of amino acid homopolymers and charge clusters typical of regulatory molecules. Nearly half of the mass of the predicted protein derives from only three amino acids: glutamine, glycine, and asparagine. Immunohistochemical studies of the protein in cell culture and early embryos show that the protein accumulates predominantly in the nucleus.
Amino Acid Sequence↗
Aerolysin of Aeromonas sobria: evidence for formation of ion-permeable channels and comparison with alpha-toxin of Staphylococcus aureus.
Aerolysin from Aeromonas sobria AB3 was isolated and purified. The pure toxin formed sodium dodecyl sulfate-insoluble oligomers in a lipidic environment. The addition of aerolysin to the aqueous phase bathing lipid bilayer membranes resulted in the formation of ion-permeable channels which had a single-channel conductance of about 70 pS in 0.1 M KCl. This defines the toxin as a channel-forming component similar to other toxins but without any indication for an association-dissociation reaction, since the channels had a long lifetime at low voltages. At voltages higher than 50 mV, the aerolysin channel switched into a closed state with a low residual conductance. The single-channel conductance was a linear function of the total aqueous conductance, which suggested that the toxin oligomers formed aqueous channels with an estimated minimal diameter of about 0.7 nm. The aerolysin pores were found to be slightly anion selective. The pore-forming properties of aerolysin were compared with those of alpha-toxin of Staphylococcus aureus. Both aerolysin and alpha-toxin share secondary structure features, must oligomerize to form pores in lipid bilayer membranes, and form channels with similar properties.
Aeromonas↗
Studies on human porin. III. Does the voltage-dependent anion channel "Porin 31HL" form part of the chloride channel complex, which is observed in different cells and thought to be affected in cystic fibrosis?
"Porin 31HL", of known primary structure, is an integral protein of the plasmalemma of human B cells (Thinnes, F.P. et al. (1989) This Journal 370, 1253-1264; Kayser, H. et al. (1989) This Journal 370, 1265-1278). Purified "Porin 31HL" from human B lymphocytes was reconstituted into lipid bilayer membranes, where it formed defined voltage-dependent channels. Five minutes preincubation with 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, potent inhibitor of chloride transport, altered the channel-forming properties of the protein, so that it now showed small irregular channels instead of distinct steps. In addition, the voltage-dependence of the channel was abolished by the action of 4,4'-diisothiocyanatostilbene-2,2'-disulfonate. Functional and structural similarities between "Porin 31HL" and porin preparations from other human tissues and from other species suggest that this protein may be part of the chloride channel complex, which is defective in cystic fibrosis.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Tumor cell invasion and gap junctional communication. I. Normal and malignant cells confronted in monolayer cultures.
Mammary tumor cells of the rat (BICR/M1Rk) and mouse (EMT6/Ro) as well as rat glioma cells (C6) are electrically coupled and show intercellular dye spreading. Monolayer cultures of synchronously beating chicken heart cells were also electrically coupled, dye spreading, however, was significantly restricted to only one or two adjacent cells. In all coupled cells, gap junctions were found in both freeze-fracture replicas and ultrathin sections. Heterologous gap junctional coupling between these tumor cells and heart cells was regularly established. The human cervix carcinoma line HeLa and the mouse L sarcoma line were electrically not coupled and did not reveal gap junctions, consequently they showed no coupling to heart cells.
Action Potentials↗
How to use Heidenhains azan staining in insects.
A newly modified, histochemical Azan staining procedure which was originally designed by Heidenhain was adapted for insects. This led to new insights in the structure of the central nervous system of flies. The somata are weakly stained with a red colored nucleus. Neuroglial regions are stained intensively red. Some undefined portions of the neuronal tracts seem to be uncolored but the unstained profiles are still visible. The other portions contain blue colored cells. These differences may be due to the cells' different monoamine content. Using this method new morphological substructures could be identified in both the mushroom bodies and the central complex of the central nervous system that do not seem to be described earlier.
Animals↗
Molecular basis of porin selectivity: membrane experiments with OmpC-PhoE and OmpF-PhoE hybrid proteins of Escherichia coli K-12.
Lipid bilayer experiments were performed with one OmpF-PhoE and several OmpC-PhoE hybrid porins of Escherichia coli K-12. All hybrid pores had approximately the same pore-forming activity, which indicated that the structure of the pores remained essentially unchanged by the genetic manipulation. This result was supported by single-channel experiments because all pores had similar single-channel conductances in potassium chloride. Measurements with other salts indicated a drastic change in the ionic selectivity when the fusion site in the ompC-phoE hybrid genes passed along the sequence of the porins from the N-terminal to the C-terminal end. Selectivity measurements using zero-current membrane potentials showed that the selectivity suddenly changed from anion to cation selectivity when a relatively short portion from the N-terminal end of PhoE was replaced by the corresponding part of OmpC. The replacement of increasing portions led to an increase in the cation selectivity until that of OmpC was reached. The change in the anion to cation selectivity is correlated with exchange of lysine-18 and serine-28 by aspartic acids. The anion selectivity of the phosphate starvation-inducible PhoE porin is closely related to the presence of several lysines spread along the primary sequence of the polypeptide chain.
Amino Acid Sequence↗
Identification of a new pore in the mitochondrial outer membrane of a porin-deficient yeast mutant.
Reconstitution experiments were performed on lipid bilayer membranes in the presence of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant and of its parent strain. The addition of the detergent-solubilized material resulted in a strong increase in the membrane conductance which was not observed if only the detergent was added to the aqueous phase. Surprisingly, the membrane conductance induced by the detergent extracts of the mutant membrane was only a factor of 20 less than that caused by the outer membrane of the parent strain under otherwise identical conditions. Single-channel recordings of lipid bilayer membranes in the presence of mitochondrial outer membranes of the yeast mutant suggested the presence of a transient pore. The reconstituted pores had a single-channel conductance of 0.21 nS in 0.1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. The pores present in the mitochondrial outer membranes of the yeast mutant shared some similarities with the pores formed by mitochondrial and bacterial porins although their effective diameter is much smaller than those of the 'normal' mitochondrial porins which have a single-channel conductance of about 0.4 nS in 0.1 M KCl, corresponding to an effective diameter of 1.7 nm. Zero-current membrane-potential measurements suggested that the second mitochondrial porin is slightly cation-selective. Its possible role in the metabolism of mitochondria is discussed.
Bacterial Outer Membrane Proteins↗
Quantum vortex dynamics in granular superconducting films.
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Journal Article↗
Influence of some instrumental parameters on the determination of quantitative data using the PET scanner PC4096-15WB.
In order to evaluate the influence of physical and instrumental limitations of the PET technique on metabolic data, various parameters have been tested. The measurements were done with a whole body PET scanner PC4096-15WB. The dependence on the transaxial resolution of reconstruction filter cut off frequency alpha c was investigated. The high resolution (4.8 mm at r = 0 in the field of view) and the low scatter fraction (less than 5%) of this system yields a higher grey/white matter ratio (3.8:1) than that found with older scanners (2:1). The use of a rotating pin source allows transmission scans to be performed after tracer administration with an error of less than 5%.
Image Processing, Computer-Assisted↗
Pores from mitochondrial outer membranes of yeast and a porin-deficient yeast mutant: a comparison.
Reconstitution experiments were performed on lipid bilayer membranes in the presence of purified mitochondrial porin from yeast and of detergent-solubilized mitochondrial outer membranes of a porin-free yeast mutant. The addition of the porin resulted in a strong increase of the membrane conductance, which was caused by the formation of ion-permeable channels in the membranes. Yeast porin has a single-channel conductance of 4.2 nS in 1 M KCl. In the open state it behaves as a general diffusion pore with an effective diameter of 1.7 nm and possesses properties similar to other mitochondrial porins. Surprisingly, the membrane conductance also increased in the presence of detergent extracts of the mitochondrial outer membrane of the mutant. Single-channel recordings of lipid bilayer membranes in the presence of small concentration of the mutant membranes suggested that this membrane also contained a pore. The reconstituted pores had a single-channel conductance of 2.0 nS in 1 M KCl and the characteristics of general diffusion pores with an estimated effective diameter of 1.2 nm. This means that the pores present in the mitochondrial outer membranes of the yeast mutant have a much smaller effective diameter than "normal" mitochondrial porins. Zero-current membrane potential measurements suggested that the second mitochondrial porin is slightly cation-selective, while yeast porin is slightly anion-selective in the open state but highly cation-selective in the closed state. The possible role of these pores in the metabolism of mitochondria is discussed.
Electric Conductivity↗