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  • 1990-1994  (11,297)
  • 1985-1989  (9,226)
  • 1970-1974  (2,638)
  • Cell & Developmental Biology  (16,821)
  • Engineering  (6,340)
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  • 101
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 169-179 
    ISSN: 0886-1544
    Keywords: calcium-binding proteins ; myonemes ; centrin ; contractility ; ciliates ; protozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Myonemes are bundles of thin filaments (3-6 nm in diameter) which mediate calcium-induced contraction of the whole or only parts of the cell body in a number of protists. In Eudiplodinium maggii, a rumen ciliate which lacks a uniform ciliation of the cell body, myonemes converge toward the bases of apical ciliary zones that can be retracted under stress conditions, entailing immobilization of the cell. An mAB (A69) has been produced that identifies a calciumbinding protein by immunoblot, immunoprecipitation experiments and specifically labels the myonemes in immunoelectron microscopy. Solubility properties, apparent molecular weight (23 kDa) and isoelectric point (4.9) of the myonemal protein, are similar to the values reported for the calcium-modulated contractile protein centrin. Western-blot analysis indicates that the 23 kDa protein crossreacts antigenically with anti-centrin antibodies. In addition, the 23 kDa protein displays calcium-induced changes in both electrophoretic and chromatographic behaviour, and contains calcium-binding domains that conform to the EF-hand structure, as known for centrin. Based on these observations, we conclude that a calcium-binding protein with major similarities to centrin occurs in the myonemes of E. maggii. We postulate that this protein plays an essential role in myonememediated retraction of the ciliature. © 1994 Wiley-Liss, Inc.
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  • 102
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 299-312 
    ISSN: 0886-1544
    Keywords: microtubule motors ; dynein ; cilia ; axoneme ; computer modeling ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: This study considers the relationship between two structural forms of the 22S dynein arm of Tetrahymena thermophila: the bouquet and the compact arm. The compact arm differs from the bouquet and from other proposed forms (e.g., the “toadstool”) in that the globular domains are situated transversely across the interdoublet gap with one globular subunit, the head, proximal to the adjacent doublet microtubule. The other models place all three globular domains proximal to the neighboring doublet microtubule. When sliding of an isolated axoneme is induced, at least 57% of total attached arms on exposed doublets are in the compact form within dimensions of 24 × 24 × 12 nm, and only about 2% of the arms are bouquets. Toadstools are incompatible with the images seen. Bouquets are not found in regions of the doublet protected by a neighboring doublet. When axonemes with exposed doublets are treated with 0.5 M KCl for 30 min, the compct arms and the dynein heavy (H)-chains disappear, while isolated bouquets and dynein H-chains appear in the medium, suggesting that the compact arms give rise to the bouquets as they are solubilized. The bouquet is the predominant form of isolated 22S dynein molecules, which are found in two apparently enantiomorphic forms, within dimensions 45 × 39 × 13 nm; bouquets attached to doublets have dimensions similar to those of isolated bouquets. Computer modeling indicates that in an intact standard-diameter axoneme, these dimensions are incompatible with the interdoublet volume available for an arm; the bouquet therefore represents an unfolded compact arm. A plausible sequence of changes can be modeled to illustrate the conversion of an attached compact arm to an attached and then free bouquet. The toadstool is probably an artifact that arises after unfolding. Consistent with the conformational difference, H-chains of attached compact arms differ from those of isolated bouquets in their susceptibility to limited proteolysis. These results suggest that the compact arm, rather than the unfolded bouquet or the toadstool, is the functional form of the outer arm in the intact axoneme. © 1994 Wiley-Liss, Inc.
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  • 103
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 350-360 
    ISSN: 0886-1544
    Keywords: microtubules ; MAPs ; cytoskeleton ; tau ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin Mr range. A high molecular weight protein of ∼290 kD that bound antibody to syncolin and to heat-stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two-dimensional SDS-PAGE revealed isolated MB proteins of pI ∼6.8 in the same molecular weight range, as well as α- and β-tubulin with pI ∼5.4. Subtilisin or high-salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by antimammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti-syncolin binding. Both syncolin and F-actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function. © 1994 Wiley-Liss, Inc.
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  • 104
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Type of Medium: Electronic Resource
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  • 105
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 59-68 
    ISSN: 0886-1544
    Keywords: cytoskeleton ; paracrystal ; coiled ribbons ; microtubule-associated proteins ; assembly ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Isolated microtubules from cod and cow brains were compared with respect to their response to calcium ions. The effect of Ca2+ on cod microtubules was found to be temperature dependent. In contrast to cow microtubules, cod microtubules assembled at 18°C. At this temperature the assembly was inhibited by Ca2+ concentrations of 2 mM and higher. This was also found for cow microtubules at 37°C. However, at 30°C there was no effect of 2 mM Ca2+ of the amount of assembly or disassembly of cod microtubules consisting of only tubulin or of tubulin and microtubule-associated proteins (MAPs). The morphology was affected though, since some coiled ribbons formed from tubulin and MAPs. The calcium-binding calmodulin did not alter the effect of calcium on cod microtubules markedly. At higher Ca2+ concentrations (〉4 mM), coiled ribbons were formed from cod tubulin and MAPs, but mainly amorphous aggregates and very few coiled ribbons were formed from cod tubulin alone, indicating that the Ca2+ effect is modulated by cod MAPs. The modulatory effect of cod MAPs was however not species specific, since both cod and cow MAPs had the same effect on cod microtubules, in spite of a different protein composition. A MAP-dependent effect of Ca2+ was also found for cow microtubule proteins. The assembly of pure cow tubulin, as well as that of cow tubulin and MAPs, was inhibited by 2 mM Ca2+. In the presence of 10 and 20 mM Ca2+, pure cow tubulin formed amorphous aggregates, rings, and even paracrystals, while the assembly of cow tubulin and MAPs was inhibited. Our results suggest therefore that the effect of Ca2+ can be moderated by MAPs, but depends on intrinsic properties of the different tubulins. © 1994 Wiley-Liss, Inc.
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  • 106
    ISSN: 0886-1544
    Keywords: kinesin ; brain mitochondria ; motility ; membrane-associated ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Kinesin, a mechanochemical enzyme that translocates membranous organelles, was initially identified and purified from soluble extracts from vertebrate brains. However, immunocytochemical and morphological approaches have demonstrated that kinesin could be associated to intracellular membranous organelles. We used an antibody raised against the head portion of the Drosophila kinesin heavy chain to reveal the presence of this protein in membranous organelles from rat brain. By using differential centrifugation and immunoblotting we observed a 116 kDa protein that crossreacts with this antibody in microsomes, synaptic vesicles, and mitochondria. This protein could be extracted from mitochondria with low salt concentrations or ATP. The 116 kDa solubilized protein has been identified as conventional kinesin based on limited sequence analysis. We also show that a polyclonal antibody raised against mitochondria-associated kinesin recognizes soluble bovine brain kinesin. The soluble and mitochondrial membrane-associated kinesins show a different isoform pattern. These results are consistent with the idea that kinesin exists as multiple isoforms that might be differentially distributed within the cell. In addition digitonin fractionation of mitochondria combined with KI extraction revealed that kinesin is a peripheral protein, preferentially located in a cholesterol-free outer membrane domain; this domain has the features of contact points between the mitochondrial outer and inner membranes. The significance of these observations on the functional regulation of the mitochondria-associated kinesin is discussed. © 1994 Wiley-Liss, Inc.
    Additional Material: 9 Ill.
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  • 107
    ISSN: 0886-1544
    Keywords: Listeria ; actin ; alpha-actinin ; vinculin ; talin ; filopodia ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: After the infectious bacterium, Listeria monocytogenes, is phagocytosed by a host cell, it leaves the lysosome and recruits the host cell's cytoskeletal proteins to assemble a stationary tail composed primarily of actin filaments cross-linked with alpha-actinin. The continual recruitment of contractile proteins to the interface between the bacterium and the tail accompanies the propulsion of the bacterium ahead of the elongating tail. When a bacterium contacts the host cell membrane, it pushes out the membrane into an undulating tubular structure or filopodium that envelops the bacterium at the tip with the tail of cytoskeletal proteins behind it. Previous work has demonstrated that alpha-actinin can be cleaved into two proteolytic fragments whose microinjection into cells interferes with stress fiber integrity. Microinjection of the 53 kD alpha-actinin fragment into cells infected with Listeria monocytogenes, induces the loss of tails from bacteria and causes the bacteria to become stationary. Infected cells that possess filopodia when injected with the 53 kD fragment lose their filopodia. These results indicate that intact alpha-actinin molecules play an important role in the intracellular motility of Listeria, presumably by stabilizing the actin fibers in the stationary tails that are required for the bacteria to move forward. Fluorescently labeled vinculin associated with the tails when it was injected into infected cells. Talin antibody staining indicated that this protein, also, is present in the tails. These observations suggest that the tails share properties of attachment plaques normally present in the host cells. This model would explain the ability of the bacterium (1) to move within the cytoplasm and (2) to push out the surface of the cell to form a filopodium. The attachment plaque proteins, alpha-actinin, talin, and vinculin, may bind and stabilize the actin filaments as they polymerize behind the bacteria and additionally could also enable the tails to bind to the cell membrane in the filopodia. © 1994 Wiley-Liss, Inc.
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  • 108
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 28 (1994), S. 179-193 
    ISSN: 0886-1544
    Keywords: acetylcholine receptor ; deep-etch replication ; sarcolemma ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We studied the organization of acetylcholine receptor (AChR) clusters by shearing cultured Xenopus muscle cells with a stream of buffer, and preparing rotary replicas of the exposed cytoplasmic surface of the sarcolemma. AChR clusters contained numerous particles that protruded from the sarcolemma and formed an irregular array composed of discrete aggregates. AChR were located within these particle aggregates, as shown by comparison of the replicas to labeling by fluorescent α-bungarotoxin, and by immunogold cytochemistry with antibodies specific for the receptor. The aggregates were cross-linked by a dense network of 7 nm filaments that replicated with the banded pattern characteristic of actin microfilaments. The organization of receptors into the small aggregates was independent of the organization of these aggregates into clusters, as alkaline extraction removed the microfilament network and disrupted the irregular array of particle aggregates, but did not disperse individual receptors from the aggregates. We conclude that two levels of interactions organize AChR clusters in Xenopus muscle cells: short-range interactions that assemble individual AChR into small aggregates, and long-range interactions, perhaps mediated by actin microfilaments, that anchor the aggregates into larger clusters. © 1994 Wiley-Liss, Inc.
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  • 109
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 72-81 
    ISSN: 0886-1544
    Keywords: spectrin ; intrinsic fluorescence ; spectrin elasticity ; fluorescence quenching ; spectrin α chain ; spectrin β chain ; membrane skeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To better understand the solution structure of spectrin, the environments of its tryptophan residues have been examined by fluorescence spectroscopy. The spectra and the extent of quenching by several quenching agents have been determined for intact spectrin and its α and β subunits. The arsenal of quenchers used in the study represented both hydrophilic and hydrophobic species including anionic, cationic and neutral compounds. Effects on spectrin fluorescence of ethanol and ionic strength, which extend and/or rigidify spectrin, and of glycerol, which is commonly used in electron microscopy of the protein, have also been assessed in the presence and absence of quenchers. Most of the tryptophans of spectrin are either internally quenched or are sequestered, hindering the approach of hydrophilic quenching agents. Both the spectral shape and the extent of quenching by acrylamide indicate that some tryptophans of the β subunit are slightly more exposed in the isolated chain than in the dimer. Similar effects on spectra and on quenching of the intact dimer and of the isolated β chain are seen when the ionic strength is reduced. Ethanol and glycerol reduce spectrin tryptophan accessibility to 2-p-toluidinyl napthalene-6-sulfonic acid (TNS). It therefore appears that low ionic strength, α-β association and neutral solute (or lowered dielectric constant) all induce a similar, but modest conformational change in the domain structure. The extent of TNS binding is not increased by lowering the ionic strength, suggesting that the expansion and/or stiffening of the molecule in low electrolyte solutions does not involve exposure of significant numbers of hydrophobic sites. © 1994 Wiley-Liss, Inc.
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  • 110
    ISSN: 0886-1544
    Keywords: intermediate filaments ; phosphorylation ; sea urchin embryos ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The effects of 6-dimethylaminopurine (6-DMAP) on the length of the cell cycle and on the state of phosphorylation of a putative intermediate filament protein, p117, have been studied in sea urchin embryos. Embryos were transferred into sea water containing 600 μM 6-DMAP at 0.5, 2 or 5 min after insemination, and incubated for 30 or 90 min. The effects of 6-DMAP on cell cycle length were studied by determining the time required for completion of mitosis upon return of the embryos in normal sea water. In all instances, except for the embryos transferred 0.5 min after insemination (AI) and incubated for 30 min, the duration of the M phase was shortened compared to controls, being faster in the embryos incubated for 90 minutes compared to the 30 min incubation period. However, embryos transferred 0.5 min AI have a longer M-phase than those transferred 2 minutes or later after fertilization, suggesting that between 0.5 and 2 min after fertilization, critical phosphorylating events occur which affect the commitment of the cells to enter M-phase.To study the pattern of p117 phosphorylation during the cell cycle, the eggs were transferred 2 minutes after fertilization in presence of 600 μM 6-DMAP and with 200 μCi/ml of 32P-orthophosphate. Analyses of 32P-labelled proteins after exposure of SDS-PAGE gels and their corresponding blots suggested that phosphorylation of p117 greatly increases at the time of pronuclear fusion, and then declines slightly at prophase-metaphase. This decrease is markedly enhanced when the cells are treated with 6-DMAP during metaphase in order to induce a premature breakdown of the mitotic apparatus. A causal link is suggested between the level of phosphorylation of p117 and its state of assembly. © 1994 Wiley-Liss, Inc.
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  • 111
    ISSN: 0886-1544
    Keywords: binding of caldesmon to myosin ; actin-activated ATPase activity of myosin ; actin-myosin interaction with in vitro motility assay ; myosin-binding domain of caldesmon ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We reported previously that smooth muscle caldesmon stimulates the ATP-de-pendent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurment and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin. © 1994 Wiley-Liss, Inc.
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  • 112
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 41-48 
    ISSN: 0886-1544
    Keywords: myosin-1 ; motility ; F-actin ; liver ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have recently purified and characterized from rat liver, polypeptides of 110-kDa and 130-kDa which possess several characteristics of myosin-1 [Coluccio and Conaty: Cell Motil. Cytoskeleton 24:189-199, 1993]. What roles these myosin-1 molecules play in hepatocytes is not yet defined. One hypothesis is that they are involved in either intracellular transport or locomotion. As a first step in establishing their function, we have investigated whether these molecules are capable of supporting motility in vitro. Our results clearly demonstrate that the isolated 130-kDa-calmodulin complex will translocate filaments at a rate of 0.03-0.05 μ/sec; motility is inhibited in free calcium ion concentrations above 0.1 μM. This inhibition is reversed with the addition of exogenous calmodulin. These results provide supporting evidence of a motile role for the 130-kDa-calmodulin complex in vivo. This is the first demonstration that in higher eukaryotes, myosin-1 from a tissue other than intestine will support motility. Partial peptide sequence analysis indicates that the 130-kDa polypeptide resembles the recently described myr 1 [Ruppert et al.: J. Cell Biol. 120:1393-1403, 1993] or MM1α [Sherr et al.: J. Cell Biol. 1405-1416, 1993] gene product. © 1994 Wiley-Liss, Inc.
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  • 113
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 79-87 
    ISSN: 0886-1544
    Keywords: microtubule transport ; microtubules ; 2,5-hexanedione ; glutaraldehyde ; kinesin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubules treated with the γ-diketone 2,5-hexanedione (2,5-HD) have altered assembly behavior characterized by precocious nucleation and rapid elongation. By measuring the rate of microtubule transport, we have examined the potential functional significance of this 2,5-HD-induced microtubule modification. 2,5-HD-treated microtubules were transported at only 70% of the rate of control microtubules in a simple kinesin-based motility assay on glass coverslips using video and computer enhanced differential interference contrast microscopy. Since 2,5-HD is capable of forming both pyrrole adducts and crosslinks with tubulin, the contributions of pyrrole formation and crosslinking to slowed microtubule transport were determined. 3-Acetyl-2,5-hexanedione (AcHD), a pyrrole forming, non-crosslinking congener of 2,5-HD which does not alter microtubule assembly, did not produce slowed microtubule transport as occurs with 2,5-HD. However, glutaraldehyde, a pyrrole-independent crosslinking agent which alters microtubule assembly in the same way as 2,5-HD, slowed microtubule transport. These results indicate that a 2,5-HD-induced microtubule modification, possibly a crosslink-related conformational change, produces both an alteration in the kinetics of assembly and an alteration in the microtubule-motor interaction. © 1994 Wiley-Liss, Inc.
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  • 114
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 375-375 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 115
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. ii 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 116
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 399-405 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: Symbolic computational systems introduce some unique features in computational engineering. There have been several papers published on the solution of differential equations under given boundary conditions by symbolic systems. The finite element formalism has received prime attention in the course of development of symbolic computation in engineering. The main idea has been to develop a symbolic FEM package to reduce the burden of manual algebra, eliminate errors introduced by numerical quadrature, and improve the efficiency of element generation.This work discusses a symbolic solution to electromagnetic linear antenna problems. The solution is a method of moments that transforms Pocklington's integral equation to a matrix equation. The symbolic system is used to produce (1) analytical integration, (2) the parametric expression for the input impedance and (3) computational code for forward and reverse problem of the input impedance.
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  • 117
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 419-432 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: This paper presents a new finite element formulation in the Laplace domain for both diffusion and wave equations with applications in the field of electrical engineering. With the aid of congruence transformation of matrices, the finite element equations in the Laplace domain are solved and time-domain results can be obtained through the inverse Laplace transform. In a test problem, good agreement between the numerical results derived with the present method and the analytical solutions has been found. For applications in which only Dirichlet and Neumann boundary conditions are involved, this new finite element approach can be applied and provide both frequency-domain and time-domain results in one run without any timestepping scheme. The limitations of using the congruence transformation in solving propagation problems are also addressed in this paper.
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  • 118
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 433-452 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: A hybrid method obtained as a combination of the coupled-mode method (CMM) and the mode-matching method (MMM) is developed and applied in the analysis of multiple dielectric and magnetic discontinuities in rectangular waveguides. As both are moment methods, some kind of truncation has to be carried out in the computer implementation. It is shown that selection of a different number of modes in the two methods is not necessary, unless low-permittivity meida inside the waveguide are considered. As a consequence, the procedure for slecting the number of basis functions is only done in one of the methods. Numerical examples are presented showing the behaviour of the method and the proofs of convergence. Examples are included illustrating the power of this hybrid technique, especially in relation to non-reciprocal structures containing magnetized ferrites.
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  • 119
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 239-252 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: Simulation results for a n+-n-n+ two-valley semiconductor device obtained by use of a shock capturing numerical agorithm are presented. The one-dimensional problem is modelled by two systems of Euler equations connected by their source terms, and the Poisson equation. The resulting system of seven equations is hyperbolic and non-linear, and it is a great problem to find an adequate numerical approximation of its time-dependent discontinuities. There are additional complications due to the stiffness of the source terms.The numerical method used in this paper is first-order acurate in time but of high spatial order in regions of smoothness. Before the method is applied, the system has to be transformed to a characteristic form. The adopted shock capturing algorithm enables the choice of the order of the accuracy by the appropriate choice of the reconstruction polynomial. Reconstructions of the second and fourth order are tested and some numerical results are presented. Because of the stiffness of the source terms, the sixth-order accurate scheme breaks down. The results presented, and especially the response obtained of the structure to the step and periodic applied voltage, prove the correctness of the used schemes.
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  • 120
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 283-304 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: An algorithm is developed to evaluate the responses of discontinuous coplanar strip-lines excited by external electromagnetic fields. The algorithm uses the cascade chain matrix method which employs the distributed circuit parameters to model the external field coupling to the line and it is applicable to most commonly encountered discontinuities in microwave integrated circuit interconnects on lossy substrates. A general CAD program is developed based on this model and it is applied to realistic coplanar strip-line interconnections with geometric and resistive discontinuities to illustrate the capabilities of the algorithm. These interconnect models are selected from a practical microwave integrated circuit design. Simplicity and fast speed of the algorithm enable computer-aided analysis of externally induced electromagnetic noise in integrated circuits to be carried out. The effects of dielectric losses in the integrated circuit substrates and the discontinuities in the conducting tracks on the wave coupling are investigated in isolation.
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  • 121
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    Chichester [u.a.] : Wiley-Blackwell
    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 329-342 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The finite element method (FEM) with local absorbing boundary conditions has been recently applied to compute electromagnetic scattering from large 3-D geometries. In this paper, we present details pertaining to code implementation and optimization. Various types of sparse matrix storage schemes are discussed and their performance is examined in terms of vectorization and net storage requirements. The system of linear equations is solved using a preconditioned biconjugate gradient (BCG) algorithm and a fairly detailed study of existing point and block preconditioners (diagonal and incomplete LU) is carried out. A modified ILU preconditioning scheme is also introducted which works better than the traditional version for our matrix systems. The parallelization of the iterative sparse solver and the matrix generation/assembly as implemented on the KSR1 multiprocessor is described and the interprocessor communication patterns are analysed in detail. Near-linear speed-up is obtained for both the iterative solver and the matrix generation/assembly phases. Results are presented for a problem having 224,476 unknowns and validated by comparison with measured data.
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 371-372 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Additional Material: 1 Tab.
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  • 123
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 1-14 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: Variational boundary element formulations are developed. Two functionals based on the dual and complementary energy approach are considered, which provide the upper and lower bound of the solution. Calculated examples of electrostatic fields are demonstrated for their field distributions and capacitances. Validity and the solution accuracy are discussed in comparison with the conventional boundary element solution.
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  • 124
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 25-33 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The method of effective penetration depth is used to produce a novel scalar or polarized finite difference approach for the solution of optical rib waveguide field problems. The new method is quicker than previous finite difference approaches and requires less computer memory. Propagation constant values are presented for a range of semiconductor rib waveguides and are found to be indistinguishable from benchmark results produced using earlier methods of analysis. A simple polarization correction formula recently derived is confirmed by utilizing the good accuracy of these results. This enables discussion of the relationship between quasi-TE, quasi-TM and scalar modes, and a further substantial reduction of the cpu time.
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  • 125
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 321-328 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The paper describes and compares the wave-chain and transmission matrix approaches to the calculation of transfer-function coefficients for ladder circuits (semi-symbolic analysis). It is found that the wave-chain matrix approach can be computationally more efficient.
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  • 126
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 343-355 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: To make possible the application of the three-dimensional finite element method to electrically large problems, it is combined with the analytical solutions of arbitrary large cavity with aperture. In this paper, detailed analysis for this hybrid method is presented. The element matrices necessary for coupling the finite element method to analytical solutions are given. The proposed hybrid method significantly reduces the number of unknown since only the inhomogeneity needs to be discretized. Thus computer memory and storage demands are reduced. In addition. This hybrid method employs the edge element which is not expected to produce spurious solutions. Also, the formulation presented in this paper preserves the sparsity of the finite element matrix, and does not require any matrix inversion. This new hybrid method is used to compute the field distributions in various partially filled rectangular enclosure. The results match well with the pure edgebased finite element method and analytical solutions.
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  • 127
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994) 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 128
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 377-398 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: Field computations by mathematical and numerical methods are presented in the time and frequency domain. The techniques used are discretization and modal expansion. The effects of modelling and frequency range on the accuracy and calculation requirements are investigated and discussed. By way of application, several results of RF-filter designs are presented, where these methods have been implemented in CAD programs.
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  • 129
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 453-453 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 130
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. i 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 131
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 75-75 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 132
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 127-139 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: In this paper the traditional square mesh 2D-TLM algorithm is generalized for rectangular meshes of arbitrary aspect ratio. It is shown that the anisotropic rectangular TLM network can be conceived in such a way that the propagation vector remains independent of the direction of propagation in the infinitesimal approximation. A full dispersion analysis of the rectangular mesh in then performed for the general case, and results are compared to that of the traditional square mesh.
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  • 133
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 155-166 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The radiation field patterns of rectangular waveguides and horns of finite dimensions are obtained numerically. The electric field integral equation is formulated to relate their radiation patterns to the surface current distribution. These currents are determined numerically by reducing the integral equation to a matrix equation, using the moment method. The computed currents are then used to calculate the radiation patterns and cross-polar fields of the finite wavegiudes and horns. The method is numerically efficient and can be applied to the computation and optimization of the antenna feed configurations.
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  • 134
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 15-24 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: This paper describes the application of the boundary element method to solving two-dimensional steady slow viscous flow problems (creeping flow) in thermal silicon oxidation. The proposed method used the velocity-pressure formulation. The use of the incompressibility condition as a boundary condition and the application of the second Green's identity to transform the domain integral into a boundary integral result in a system of three boundary integral equations for velocity components and pressure. Solution of this system to be an ill-posed problem because of the presence of boundary conditions of the first kind. Two methods of regularization are employed. The numerical results for trench oxidation process are presented.
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  • 135
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994) 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 136
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 43-67 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The particle-in-cell method is coupled with boundary-fitted co-ordinates in order to model the stationary Maxwell-Lorentz problem in technical devices. New numerical algorithms describing the transition between the grid model and the mesh-free model are developed and the existing techniques of finite difference schemes for equidistant grids are extended to non-equidistant, arbitrarily shaped, convex four-point meshes. The modelling process is described, and both the numerical approximation and the algorithms are discussed. Applications in different technical devices show the flexibility of the method.
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  • 137
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 77-84 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: Series function expansion algorithms of the time-domain analysis of boundary value problems are discussed. Electromagnetic fields inside a structure under investigation are expanded into series of basis functions and the expansion coefficients are found by means of the Galerkin method. The numerical cost of algorithms is discussed and a cost efficient approach is proposed for formulations using sine and cosine expansion functions. Compared with conventional time-domain methods the algorithms described show the time evolution of the expansion coefficients rather than the samples of a physical continuum at discrete nodes.
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  • 138
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 69-74 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The application of multigrid techniques to the computation of the static solutions of electromagnetic field problems governed by Laplace's equation is described. This technique is compared with the conventional successive over-relaxation (SOR) method for solving finite difference problems. In contrast to SOR, the number of iterations of multigrid needed to achieve convergence is largely independent of the grid size. It is shown that the relative performance of multigrid is excellent on large grids where the number of iterations of SOR needed to achieve convergence becomes prohibitively large. The technique is illustrated by applying a parallel implementation of multigrid to find a quasistatic solution of a boxed microstrip problem.
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  • 139
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 85-97 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: A new approach to efficiently compute the low-frequency eigenmodes of a time-domain model approximating a linear physical system is proposed. The method is based on principles known from digital signal processing, in particular from parametic spectrum estimation, so it is not surprising that the achievable accuracy is much higher than the accuracy of the usual non-parametric discrete Fourier transform approach. The algorithm works in the general lossy case even for a very large number of unknowns and can easily be extended to calculate steady-state solutions under sinusoidal excitations for several different frequencies simultaneously.
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  • 140
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 35-41 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The eigenmodes in electromagnetic cavities, loaded with arbitrarily shaped dielectric materials, are computed by the edge element method. The computation shows that the well known ‘spurious modes’ no longer appear. Formulae are given and verified so that the number of zero eigenvalues, which come from ∇ × H = 0, may be accurately predicted. Formulae for the order and density of the global matrices are given for a rectangular cavity regularly divided into bricks and tetrahedra, allowing an associated comparison between the contrasting edge and nodal element formulations. Comparison of the computed results with available theoretical and previously published data show the edge element approach to be a robust, accurate and effective method.
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  • 141
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 99-125 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: In this paper we demonstrate the use of differential equations by means of an example from network analysis and show that differential/algebraic equations (DAE), rather than explicit ordinary differential equations (ODE), are more suitable for the description of electrical systems and networks. The main ideas of numerical integration of ODEs are presented. We consider this material from the point of view of replacing the ODE by a difference equation (DE). In particular, the relationship between the ODE and the associated DE is discussed. In the last Section the application of integration methods for OEDs upon DAEs and its difficulties are discussed. The paper is intended as a review; but a few new results are also included.
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  • 142
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994) 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 143
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 141-153 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: Applying the method of moments to Maxwell's equations, Yee's two-dimensional FDTD scheme with central difference approximations and the two-dimensional TLM method are dervied from first principles of field theory. By comparing the eigenvalues of the two methods, the differences between two-dimensional FDTD and TLM are illustrated.
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  • 144
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 179-188 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: A convex hexahedral TLM mesh of arbitrary shape is presented and the transmission-line matrix method extended to any non-orthogonal configuration. The novel mesh constitutes a natural generalization of Johns' condensed node. The associated TLM process is analysed and reconstructed as a genuine finite difference time-domain algorithm. Nodal S-parameters are derived from discretized Maxwell's equations and canonical stability criteria yield the TLM timestep. Unitarity is discussed and energy conservation confirmed in the non-conductive case.A given block-diagonal representation of the S-matrix restrains processing time per node and iteration within the range of traditional methods. The shortcomings of the rigid classical grid, as the need for inaccurate staircasing approximations, are, however, ruled out.Our analysis takes advantage of the recently developed propagator integral approach.
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  • 145
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 201-207 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The existing treatment of an instantaneous endothermic change of state is extended to deal both with exothermic effects and with first order Arrhenius changes. These extended treatments are used to model complex changes of phase occurring during the firing of ball clay and china clay, both of which are constitutents of the slip used in the production of vitreous china ware. The adequacy of the treatments is investigated for both clays by comparing a TLM simulation of a DSC (differential scanning calorimetry) experiment with measurements made during the experiment.
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  • 146
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 225-238 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: Transverse transmission concepts in the Fourier transmission domain for multilayer planar transmission medium are presented. This complete transverse transmission theory (TTT) is found to be a superset of different Fourier-transform-based numerical techniques widely used in guided wave analysis, namely the modematching method and the spectral-domain approach. The features of the theory are: the expansion of field quantities in longitudinal section (LSE/LSM) modes, the concept of rotation of the transverse field vectors, and a comprehnsive algorithm using recursive matrix standard form. It is shown that the mode-matching method and spectral-domain approach are generally equivalent except for the last step of imposing the final boundary conditions, and can therefore be summarized in a unified theory within the framework of TTT. In addition, a new parameter, the polarization coefficient, has been introduced to describe the field polarization of the guided wave in a quantitative manner. It is believed that the present theory will help us to gain more insight into hybrid mode propagation in multilayer planar circuits.
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  • 147
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 267-281 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: This paper considers the adaptation of drift-diffusion device simulation methodology to study Auger-recombination-induced hot electron transport characteristics in InGaAsP/InP double heterostructure laser diodes. In order to model the transport behaviour of the Auger hot electrons, we decompose the conventional electron current continuity equation into two components, with one for the Auger hot electrons and the other for the low-energy electrons. These equations, which use the energy relaxation time parameter to model the dynamics of the Auger hot electrons, are then coupled with the hole current continuity equation and the Poisson equation to obtain self-consistent solutions. Results from the case studies of one-dimensional N-p-P InGaAsP/InP double heterojunction laser diodes with material composition corresponding to 1·3 μm and 1·55 μm wavelength emissions are presented. We have observed that hot electrons generated through Auger recombination inside the active region can spread into both the N- and the P-InP cladding layers. Within the drift-diffusion framework, it is demonstrated that the hot electron concentration in the N-InP cladding layer can be five orders of magnitude higher than that in the P-InP cladding layer. Because energy transport of the hot electrons in not modelled under the drift-diffusion approximation, the simulated results are discussed to highlight some of the possible limitations in using drift-diffusion physics to study Auger hot electron transport behaviour. The importance of taking energy transport into account is emphasized.
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 307-307 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 149
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 167-177 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: Ray-besed methods have been previously developed for accurately predicting the electromagnetic scattering from cavity interiors that allowed for realistic modelling of complex cavities such as jet engine inlets. An analytic geometric model is developed for bifurcated cavities based on connecting sections of generalized super-ellipse functions. The electromagnetic scattering from such cavities using the ‘shooting and bouncing rays’ (SBR) method is discussed.
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  • 150
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 209-221 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: A theoretical analysis has been developed to study the crosstalk in crossed bundles of parallel lines. The model is based on the theory of the coupled transmission lines and the resulting system of differential equations has been solved by means of the finite difference, time-domain (FDTD) method. The frequency response of the network has been achieved by the discrete fourier transform (DFT) algorithm; measurements, performed with a vectorial network analyser, show a good agreement with numerical simulation.
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  • 151
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 253-265 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: A numerical frequence-domain modelling of two-terminal, non-linear microwave circuits is presented. It basically relies on a process allowing the solution of the frequency-domain curcuit harmonic balance equations while accounting for the semiconductor device by means of an accurate numerical macroscopic physical model. In its present state of development, the model allows the study of a single two-terminal device circuit operating in harmonic mode. Its capabilites are illustrated by means of the results of a study devoted to the optimization of the load curcuit configuration of a millimetre-wave avalanche diode frequency multiplier. The influence of the output load impedance level on the circuit output RF performance has been investigated for different input power levels in direct frequency multiplication mode and in the presence of additional circuit tunings at low harmonic rank idler frequencies.
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  • 152
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994) 
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    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 153
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 309-319 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: This paper describes a rigorous and systematic procedure to derive a non-linear distributed FET model that an easily be implemented in CAD routines of simulators based on harmonic balance techniques. The new model is derived from a knowldge of the conventional linear lumped equivalent circuit, from non-linear current sources extracted with pulsed measurements, and from the physical dimensions of the FET.For fundamental and haromonic requencies, the FET is modelled by N identical cells. Each cell is made up of a non-linear two-port section inserted between two linear four-port sections that simulate the coupling and the distributed effects along the electrodes of the FET in the width direction only. This non-linear distributed scaling approach to FET modelling has been applied to the analysis of a submicrometre-gate GaAs FET at Millimetre-wave frequencies, and the results were compared to the non-linear lumped element approach. This approach can be applied to other transistors used in non-linear regions at microwave and millimetre-wave frequencies.
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994) 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 155
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. ii 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 156
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 189-199 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: A rigorous technique for studying a thin wire circular loop antenna excited by a magnetic frill is developed. The exact kernel is derived and used to improve the accuracy. The scheme is applicable to small as well as large loops. Results obtained are nearly indistinguishable from those determined by the Fourier expansion method for non-resonant loops. A disagreement of 2 per cent is generally observed when the loop is close to reasonant for βb 〉 2. Compared with the conventional numerical method where a loop is represented by a polygon of straight wires, the present scheme is much faster and more accurate. Extension of this method to cover an antenna system of arbitrarily oriented loops intermingled with straight wires can be easily achieved.
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  • 157
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994) 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
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  • 158
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 357-370 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: The calculation of potentials in homogeneous and isotropic media by the boundary element method has the advantage that a harmonic solution of Laplace's equation is obtained for given approximated boundary conditions. The technique leads to the solution of linear systems with full matrices of dimension 1000-10,000 for medium- and large-sized three-dimensional problems, An efficient solution procedure of the linear systems is required.While the iterative solution of the large and sparse linear systems arising from the finite difference or the finite element method is well documented, the systems resulting from the boundary element method are typically solved by direct methods. However, in many cases an iterative solver needs far fewer operations to achieve a sufficient accuracy. Importantly, there are many alternative methods, each of them well suited for different types of problem.Here, we provide an overview of state-of-the-art iterative solvers. We will discuss the particular methods that have been successfully applied to systems arising from field calculations in the high-voltage engineering by the boundary element method. The selection of appropriate methods is discussed. We demonstrate that iterative solutions can be much faster than direct solvers with regards to the number of operations. Furthermore, these solvers are optimally suited for today's supercomputers because they can be efficiently vectorized and parallelized.
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  • 159
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    International Journal of Numerical Modelling: Electronic Networks, Devices and Fields 7 (1994), S. 407-418 
    ISSN: 0894-3370
    Keywords: Engineering ; Electrical and Electronics Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Electrical Engineering, Measurement and Control Technology
    Notes: This paper provides details of a widely applicable technique of hybrid modelling of thermal and particle diffusion which can help to reduce computational load in explicit formulation. The undesirable effects of artificial boundaries, which are introduced when a simulation is truncated or when an expanding mesh is used, can be eliminated by the use of discrete Green's functions. The technique can also be applied to problems involving infinite or near-infinite domains or problems where a complex thermal region is embedded within a larger, more simply described domain.
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  • 160
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    Molecular Reproduction and Development 37 (1994), S. 61-68 
    ISSN: 1040-452X
    Keywords: Aging oocytes ; Pronuclei ; Cattle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Bovine oocytes matured in vitro (IVM) for 20 hr vs. 40 hr were treated for activation with 7% ethanol in Dulbecco's phosphate-buffered saline for 5 min followed by incubation in M199 + 7.5% fetal calf serum containing cycloheximide (10 μg/ml). TreatedIVM oocytes and the controls (no ethanol and cycloheximide exposures) were fixed after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation and stained 24 hr later with 1% acetoorcein to examine nuclear events. Different stages of nuclear development of the activated oocytes were identified on the basis of nuclear and chromosomal morphology. Pronuclear development was classified into four stages (PN I, II, III, and IV) according to pronuclear progression in chromatin decondensation, nucleoplasm appearance, and nuclear size. The results demonstrated that the combined activation treatment effectively drove the IVM oocytes, both young (20 hr) and aging (40 hr), out of metaphase arrest. The activation rates for young oocytes examined immediately after 0, 1, 2, 3, 4, 5, 7, 10, and 20 hr of incubation with cycloheximide were, respectively, 7%, 24%, 77%, 96%, 92%, 97%, 98%, 93%, and 98%. For aging oocytes (40 hr) the corresponding activation values at the same time intervals were 6%, 84%, 100%, 100%, 100%, 100%, 98%, 100%, and 100%, respectively. These values were significantly higher than those for the corresponding controls. The activated aging oocytes achieved peak activation response more rapidly than did young oocytes. In addition, nuclear events in aging oocytes proceeded faster than those in young ones. Spontaneous activation rates of the aging oocytes were also higher (6-57%) than those of the young ones (0-14%). © 1994 Wiley-Liss, Inc.
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  • 161
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    Molecular Reproduction and Development 37 (1994), S. 12-20 
    ISSN: 1040-452X
    Keywords: Spermatids ; Translational regulation ; mRNPs ; Translational initiation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Protamine 1 mRNAs are inactivated by a block to the initiation of translation in early spermatids and are translationally active in late spermatids in mice. To determine whether translation of protamine 1 mRNAs is inhibited by a protein repressor, the translational activity of ribonucleoprotein particles and deproteinized RNAs were compared in the reticulocyte and wheat germ cell-free translation lysates. To isolate RNPs, cytoplasmic extracts of total testes were fractionated by large-pore gel filtration chromatography. Ribonucleoprotein particles in the excluded fractions stimulated synthesis of radiolabeled translation products for protamine 1 about twofold less effectively than deproteinized RNAs in the reticulocyte lysate, but were inactive in the wheat germ lysate. The ability of translationally repressed protamine 1 ribonucleoprotein particles to form initiation complexes with 80S ribosomes in the reticulocyte lysate was also measured. Protamine 1 ribonucleoprotein particles isolated by gel filtration and in unfractionated cytoplasmic extracts of early spermatids were nearly as active in forming initiation complexes as deproteinized mRNAs. The isolation of ribonucleoprotein particles in buffers of varying ionic strength, protease inhibitors, and several other variables had no major effect on the ability of protamine 1 ribonucleoprotein particles to form initiation complexes in the reticulocyte lysate. These results can be explained by artifacts in the isolation or assay of ribonucleoprotein particles or by postulating that protamine 1 mRNAs are inactivated by a mechanism that does not involve protein repressors, such as sequestration. © 1994 Wiley-Liss, Inc.
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  • 162
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    Molecular Reproduction and Development 37 (1994), S. 54-60 
    ISSN: 1040-452X
    Keywords: Cattle ; Cumulus oophorus ; Folicular fluid ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Since bovine cumulus oophorous and oviductual cell cultures are known to support and maintain frozen-thawed bovine sperm viability and motility for extended time periods, we investigated whether granulosa cell (GC)- and oviductual cell (OC)-conditioned media have similar effects. GC and OC were cultured for 3 days in TCM-199 medium supplemented with 10% fetal calf serum. At that time, the supernatant was discarded from GC and the monolayers were covered with Sp-TALP medium containing 6 mg/ml bovine serum albumin, while the OC were recovered by centrifugation and transferred to culture bottles containing Sp-TALP. Two days later, GC-conditioned and OC-conditioned Sp-TALP were recovered and dialyzed, and their retentates were lyophilized. Bovine follicular fluid (BFF) was also dialyzed, and its retentate was lyophilized. When sperm were incubated in GC- or OC-conditioned media, motility remained above 62% and 42% at 6 hr and 30 hr, respectively, and motility was higher than that of the control both at 6 hr (39%; P 〈 0.001) and at 30 hr (9%; P 〈 0.0001). Similarly, when sperm were incubated in the lyophilized retentates of GC- and OC-conditioned media and in BFF at a dose of 0.1, 0.5, or 1.0 mg/ml, the motility rates were higher both at 6 hr (P 〈 0.05) and at 30 hr (P 〈 0.01) compared to the control. The increase in motility was dose dependent; a 1.0 mg/ml dose improved (P 〈 0.05) motility compared to a 0.1 mg mg/ml dose. Heat treatment of the retentates of GC, OC, and BFF at 55°C for 30 min did not destroy their ability to support and maintain motility. However, heating at 100°C for 5 min destroyed their ability to support motility. Molecular sieving of retentates on Sephancryl S-300 yielded fractions that were highly effective (P 〈 0.01) in enhancing and maintaining motility compared to the other fractions. In conclusion, GC and OC secrete nondialyzable, heat-labile factor(s), which support and maintain sperm viability and motility for up to 30 hr. © 1994 Wiley-Liss, Inc.
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  • 163
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    Molecular Reproduction and Development 37 (1994), S. 93-98 
    ISSN: 1040-452X
    Keywords: Chromatin condensation ; DNA packaging ; Spermatozoa ; Hamster ; Acridine orange ; Flow cytometry ; Bromobimane ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this study we have used acridine orange staining, as described by Evenson (1990), to follow changes in DNA packaging as they occur in hamster spermatozoa which have left the testis and are undergoing maturation in the epididymis. Measurement of the green and red fluorescent intensities of hamster sperm nuclei by flow cytometry demonstrated a decrease in acridine orange binding to DNA as sperm made their way from proximal corpus epididymis to the vas deferens. Using sperm from the cauda epididymis of the mature hamster as the standard, a method was developed for estimating the % of cells in a given sample that have matured with regard to DNA packaging. Staining with bromobimane was used to determine the extent of sulfhydryl oxidation in the nuclei. It was seen that sulfhydryl oxidation occurred mainly in the cauda epididymis whereas another process in chromatin condensation occurred earlier, during sperm passage through the caput epididymis. This earlier process could be mimicked by incubating sperm nuclei with alkaline phosphatase, suggesting that it consists of removal of phosphate in protamine. © 1994 Wiley-Liss, Inc.
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  • 164
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    Molecular Reproduction and Development 37 (1994), S. 99-109 
    ISSN: 1040-452X
    Keywords: Oocyte growth ; Oocyte maturation ; Egg ; Actin ; Myosin ; Spectrin ; Animal-vegetal polarity ; Cortex ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The distribution of contractile proteins, actin and myosin, and an actin-binding protein, spectrin, was studied in oogenesis of Xenopus laevis. These proteins are present in oocytes already at the previtellogenic stages, which are characterized by their diffuse distribution. The localization of proteins changed with the beginning of vitellogenesis. At all vitellogenic stages, including the fully grown oocyte, animal-vegetal differences were noted in localization of actin and myosin: in the animal hemisphere they appear as fibrillar-like structures, while in the vegetal one they are localized around the yolk platelets. By the end of the oocyte's growth, a cortical gradient appeared: predominant localization of actin and myosin in the cortical area. As the oocyte maturation proceeded, the distribution of actin and myosin again became diffuse and nonuniform, so that a cortical gradient appears. At the beginning of vitellogenesis spectrin is distributed as a network all over the ooplasm, while in the fully grown oocyte it is localized mostly in teh subcortical area of the animal hemisphere and, as individual inclusions, in other regions of the oocyte. No spectrin is found by the end of maturation. Actin, myosin, and spectrin are also present in the oocyte's nuclei. Changes in the distribution of contractile proteins and spectrin during oocyte maturation are discussed with respect to the development of cortical contractility, as well as to the changes in spatial distribution of yolk platelets and regional sensitivity of the maturing oocyte to cytochalasin B. © 1994 Wiley-Liss, Inc.
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  • 165
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    Molecular Reproduction and Development 37 (1994), S. 110-119 
    ISSN: 1040-452X
    Keywords: Sperm ; Glycoprotein ; GPI ; Maturation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The principal galactose oxidase/NaB[3H]4-labeled membrane protein of rat caudal epididymal spermatozoa was isolated by hydrophobic interaction chromatography. The protein is released from the membrane by the action of phosphatidylinositol specific phospholipase C, and thereby its properties are transformed from those of a protein anchored to the hydrophobic membrane to those of a hydrophilic solution protein. Because it is the only membrane-associated protein released by the enzyme which did not absorb to a propylaspartate resin, a simple, single step purification procedure was devised.Although the amino terminus of the protein is blocked to Edman degradation, the majority of the protein structure was determined from a series of tryptic peptides and from limited acid hydrolysis. Approximately 65% of the protein mass is carbohydrate which is primarily attached through O-glycosidic bonds to the 18 threonines. The molecular weight of the glycoprotein was estimated to be 16,600, considerably smaller than the Mr = 26,000 to 37,000 previously determined by gel electrophoresis. The anomalous electrophoretic behavior is undoubtedly due to the large percentage of carbohydrate. The distribution of carbohydrate on the protein side chains suggests the protein may form a positively charged, specialized scaffolding for the presentation of the carbohydrate moieties. Because the appearance of the ability to label the protein with galactose oxidase is correlated with sperm maturation in the epididymis, the glycoprotein structures may be an important componetn in the fertilization process. The combination of linkage by glycosylphosphatidylinositol and low molecular weight mucin-like structure indicates this may be a member of a new class of membrane proteins. © 1994 Wiley-Liss, Inc.
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  • 166
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    Molecular Reproduction and Development 37 (1994) 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 167
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    Molecular Reproduction and Development 37 (1994), S. 120-120 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 168
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    Molecular Reproduction and Development 37 (1994), S. 146-156 
    ISSN: 1040-452X
    Keywords: Fertilization ; Sperm Remodelling ; Oocyte Fragments ; Cybrids ; Mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Nucleate and anucleate fragments of parthenogenetically activated mouse oocytes, as well as cybrids obtained by fusion of anucleate fragments (cytoplasts) of maturing and activated matured oocytes were fertilized at different time after activation. Remodelling of the sperm nucleus was studied by electron microscopy at 1.5 and 3 h after fertilization and, in addition, at 14 h in cybrids. Results show that (1) the nuclear envelope of the sperm nucleus can break down when the insemination takes place after the end of M-phase, but the capacity of the parthenote cytoplasm to remodel the sperm nucleus is restricted in time. (2) Male chromatin can decondense within the old, unbroken nuclear envelope, but in such cases formation of a male pronucleus, one of the two nuclei of zygote possessing inactive nucleoli, is never observed. © 1994 Wiley-Liss, Inc.
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  • 169
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    Molecular Reproduction and Development 37 (1994), S. 130-137 
    ISSN: 1040-452X
    Keywords: Human Epididymis ; HE genes ; Gene family ; Pseudogene ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Differential screening of a human epididymal cDNA library led to the isolation and characterization of a major epididymis-specific cDNA clone family, referred to as HE3. More detailed sequence and PCR analysis identified two different but homologous gene transcripts, HE3α and HE3β. The former represents an mRNA of ca. 1 kb, encoding a putative small secretory polypeptide of 14903 MW. The HE3β transcript was only found as incomplete 3′ fragments. Analysis of human genomic DNA by Southern blotting suggested the presence in the human genome of at least three independent HE3-related genes. Isolation of genomic clones for the HE3α gene showed this to contain a single intron of 1.4 kb in the 5′ noncoding region. Although genomic clones corresponding to HE3β could not be found, a third highly homologous gene, HE3γ, was identified as a potential pseudogene. Neither nucleotide nor encoded amino acid sequences of the HE3 gene family are related to any other known sequence in the central databases, and thus represents a novel human gene family, with at least three nonallelic members. Northern hybridization analysis showed that HE3 gene products are specifically expressed in the human epididymis, and not in any other tissue examined. Furthermore, except for the pig, no other nonprimate species has been identified to express homologous sequences in the epididymis. RNase protection assays showed that both the HE3α and HE3β, but not the HE3γ genes, are expressed in the human epididymis. © 1994 Wiley-Liss, Inc.
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  • 170
    ISSN: 1040-452X
    Keywords: Neurotrophin receptors ; Testis development ; Spermatogenesis ; Male germ cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Nerve growth factor (NGF) is essential for neuronal development and differentiation. Recent reports have shown that its low-affinity receptor (LNGFR) is expressed and developmentally regulated in a broad range of embryonic and adult tissues outside the nervous system, although the functions of the receptor in such tissues remain unknown. Recently, NGF and LNGFR have been detected in adult mouse, rat, and human testis.The results of the present work demonstrate that LNGFR is expressed much before the onset of spermatogenesis in both mouse and rat testis. In situ hybridization shows that the mRNA for LNGFR is expressed in the peritubular cells of the embryonic mouse testis. Immunohistochemical analysis of the rat testis shows LNGFR-expressing cells to be scattered in the intertubular compartment in the embryonic testis, and to become organized in a cellular layer that surrounds myoid cells of the seminiferous tubules during postnatal development. Furthermore, in peripuberal and adult mouse and rat testis we have identified the expression of an abundant and shorter mRNA of 3.2 kb that cross hybridizes to the low-affinity NGF receptor transcript (3.7 kb). This shorter mRNA species, which appears at the beginning of spermatogenesis in the adult, has been identified by in situ hybridization and by Northern blot with RNA isolated from homogeneous populations of meiotic germ cells to be expressed by pachytene spermatocytes and round spermatids. Our results suggest a complex developmental role for LNGFR during testicular morphogenesis and identify the expression, at specific stages of spermatogenesis, of a new germ cell - specific transcript homologous to the receptor RNA. © 1994 Wiiey-Liss, inc.
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  • 171
    ISSN: 1040-452X
    Keywords: Acrosin ; Acrosome reaction ; Rabbit sperm ; Perivitelline spermatozoa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The participation of acrosin in mammalian sperm penetration through the zona pellucida has been amply debated. In this paper we report the immunolocalization - by silver enhanced immunogold technique using ACRO-8C10 monoclonal antibody to human acrosin - of proacrosin/acrosin on ejaculated rabbit spermatozoa incubated in vitro in a capacitating medium and on spermatozoa recovered from the perivitelline space. After incubation in a capacitating medium, four different patterns were observed: (1) no labeling on acrosome intact spermatozoa; (2) labeling on the rim of the head; (3) labeling on the whole acrosome area; and (4) no labeling on acrosome reacted spermatozoa. At the start of incubation, spermatozoa with pattern 1 were the most abundant, whereas at the end of the 32 h incubation period, patterns 2 and 3 were the most frequent. On the other hand, 625 perivitelline spermatozoa were recovered from 17 fertilized rabbit eggs, of which 26% were labeled with the anti-acrosin monoclonal antibody ACRO-8C10 in two different areas: (1) only on the equatorial region; and (2) only on the postacrosomal area. These results are consistent with the idea that proacrosin/acrosin remains associated to the acrosome reacted spermatozoa for long periods of time, and that proacrosin/acrosin associated to perivitelline spermatozoa could be responsible for the second penetration of fresh rabbit eggs by perivitelline spermatozoa. © 1994 Wiley-Liss, Inc.
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  • 172
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    Molecular Reproduction and Development 37 (1994), S. 241-246 
    ISSN: 1040-452X
    Keywords: Spermatozoa ; semen preservation ; Cytosolic calcium ; Fura-2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The intracellular free calcium concentration [Ca2+]i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 μM progesterone so that the regulation of [Ca2+]i in a dynamic situation could be studied. [Ca2+]i (nM) was 290 ± 13 in fresh spermatozoa vs. 550 ± 26 in cryopreserved samples (mean ± S.E.M. P 〈 0.0001 paired t-test). Progesterone at a dose of 3.18 μM stimulated a large and rapid increase in [Ca2+]i to a peak value 〉 1 μM after 10-20 seconds. [Ca2+]i then declined to a slightly raised basal level over the next 30-40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca2+] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 μM) completely inhibited the transient rise in [Ca2+]i produced by progesterone, but 100 μM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca2+]i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation. © 1994 Wiley-Liss, Inc.
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  • 173
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    Molecular Reproduction and Development 37 (1994), S. 255-263 
    ISSN: 1040-452X
    Keywords: Phosphorylation ; Chromatin condensation ; Testis ; DNA binding proteins ; Seminiferous tubules ; Sonication-resistant nuclei ; mP1 ; Pre-mP2 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mouse and rat seminiferous tubule fragment cultures were used to examine synthesis and processing of mammalian protamines and transition proteins. The tubule fragments were incubated with [3H]-arginine, [3H]-histidine, [35S]-cysteine, or [32P]-PO4, and radiolabeled proteins were analyzed by acid/urea polyacrylamide gel electrophoresis and fluorography or autoradiography. Newly synthesized protamines were recovered from sonication-resistant nuclei (SRN) and could not be detected in cytoplasmic fractions, indicating that protamines are deposited into nuclei immediately after synthesis. Newly synthesized mouse protamine 1 (mP1) and the precursor to mouse protamine 2 (pre-mP2) migrated more slowly during electrophoresis than their predominant testicular forms, identified by staining with Coomassie blue R-250. Within 1 hour of synthesis, the electrophoretic mobilities of mP1 and pre-mP2 increased to match those of their predominant forms. These changes are consistent with initial charge-neutralizing modifications of the newly synthesized protamines, followed by removal of at least some of the modifying ligands, to unmask protamine basicity. Steady-state phosphorylation rates were high for rat protamine 1 (rP1) and were independent of phosphate content; both rP1 molecules of low and high phosphate content were rapidly phosphorylated. Pre-mP2-3, a major processing intermediate derived by proteolysis of pre-mP2, was also rapidly phosphorylated. Like the protamines, transition protein 2 (TP2) was rapidly phosphorylated and increased in electrophoretic mobility soon after synthesis. In contrast, transition protein 1 (TP1) was not phosphorylated and did not exhibit multiple electrophoretic forms. © 1994 Wiley-Liss, Inc.
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  • 174
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    Molecular Reproduction and Development 37 (1994), S. 276-283 
    ISSN: 1040-452X
    Keywords: Milk proteins ; Mammary gland ; PCR ; Microinjection ; RIA ; Expression ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Fragments containing 5′ flanking regions of four bovine milk protein genes - alpha lactalbumin (bαLA), alpha S1 casein (bαS1CN), beta casein (bβCN), kappa casein (bkCN) - and mouse whey acidic protein (mWAP) gene were prepared by PCR and ligated to human growth hormone (hGH) gene. These recombinant DNAs were microinjected into rat embryos to produce transgenic rats, and the functions of the 5′ regions to direct secretion of hGH in the milk were tested. Although milk was obtained only in 5 of 19 mWAP/hGH rat lines, more than two-thirds of the rats carrying the other four DNAs produced milk. More than 80% of the lactated rats carrying bαLA/, bβCN/, and mWAP/hGH, and 33% of the laclated bαS1CN/hGH rats secreted detectable amounts of hGH (〉 0.05 μg/ml) in the milk. In some rats, the hGH concentrations in the milk were comparable to or more than that of the corresponding milk protein in bovine milk. The ranges of hGH concentrations in the milk of bαLA/, bβCN/, bαS1CN/, and mWAP/hGH rats were 1.13-4,360 μg/ml, 0.11-10,900 μg/ml, 86.8-6,480 μg/ml, and 6.87-151 μg/ml, respectively. HGH was also detected in the sera of these rats, and some abnormalities of growth and reproduction were observed. All but one virgin mWAP/hGH rat secreted up to 0.0722 μg/ml of hGH in the serum, and more than half of them showed abnormal fat accumulations at their abdomen. None of the bαCN/hGH rats secreted detectable amount of hGH into their milk, whereas 8 of the 11 lines secreted hGH into their sera. For the production of hGH in transgenic rat milk, the 5′ region of bαS1CN was shown most suitable, because the bαS1CN/hGH rat secreted 〉 6,000 μg/ml of hGH into the milk and could be reproduced. © 1994 Wiley-Liss, Inc.
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  • 175
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    Molecular Reproduction and Development 37 (1994), S. 305-317 
    ISSN: 1040-452X
    Keywords: HeLa extracts ; Chromatin decondensation ; Remodelling ; Recondensation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: HeLa cell extracts induced decondensation of lysolecithin permeabilized Xenopus, pig, and human sperm chromatin; decondensation began almost immediately on incubation in the extract and was completed within 10-20 min. The average enlargements of human and pig sperm nuclei were 15-fold and 3-fold, respectively. The structural organization of pig and human sperm chromatin was significantly differnt. Decondensation was differentially inhibited by Mg++ and polyamines; inhibition was least for Xenopus and most for pig sperm nuclei. The nuclear membrane was disintegrated on chromatin dispersion, whereas the nuclei which failed to decondense exhibited distinct nuclear envelopes. The decondensing factors were stable at 65°C for 15 min. The dispersed chromatin was remodelled to somatic nucleosomal structures within 60 min. The remodelled chromatin could be recondensed to chromosome-like structures, when incubated further in extracts from mitosis arrested HeLa cells. © 1994 Wiley-Liss, Inc.
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  • 176
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    Molecular Reproduction and Development 37 (1994) 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 177
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    Molecular Reproduction and Development 37 (1994), S. 370-381 
    ISSN: 1040-452X
    Keywords: Sex determination ; Sex determining region Y ; Postmeiotic expression ; HMG box containing proteins ; Interstitial cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Although its expression in adult testis was immediately apparent, the role for Sry (sex determining region, Y) in testicular function remains elusive. We have performed transcriptional studies in an effort to elucidate potential roles of Sry by studying the time and location of its transcription in mouse testes. Northern analyses and more sensitive nuclease protection assays detected transcripts in 28-day-old testes and beyond. The highly sensitive technique of reverse transcription polymerase chain reaction (RTPCR) could not detect Sry expression in 14-day testes when primers for the most conserved portion of the gene, the high mobility group (HMG) box, were used, but primers for the circular form detected Sry transcription at all postnatal stages studied. The same HMG box primers were able to detect expression of Sry in XX, Sxra or Sxrb testes. This suggested that Sry is expressed in cells other than germ cells, which was confirmed with studies on fractionated cells - RTPCR detected transcription of Sry in the highly pure interstitial cell fraction. However, Leydig cells and a Leydig cell tumor were negative for Sry expression. We performed in situ studies in an attempt to localize the expression of Sry in the testes. Abundant expression of an Sry cross-hybridizing transcript was found in spermatogonia, in early spermatocytes, and in some interstitial cells with antisense probes to the HMG box or a more specific, 3′ region, whereas the sense probe gave little or no hybridization. It is probable that the circular transcripts, which are seen in reverse transcriptase positive (RT+) and RT- reactions by PCR because of the RT activity of Taq polymerase, are responsible for the hybridization seen in spermatogonia and spermatocytes, whereas linear and circular forms are detected later. Thus Sry is expressed in pre- and postmeiotic germ cells and in somatic cells of the testes. © 1994 Wiley-Liss, Inc.
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  • 178
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    Molecular Reproduction and Development 37 (1994), S. 382-390 
    ISSN: 1040-452X
    Keywords: Insulin-like growth factors ; Diabetes ; Embryos ; Mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Insulin-like growth factors (IGF-I and IGF-II) play an important regulatory role in fetal growth and development. Alterations in expression of these growth factors may result in developmental abnormalities, macrosomia, and intrauterine growth retardation, which occur with a higher incidence in diabetic pregnancies. In situ hybridization histochemistry was employed to investigate the distribution and abundance of IGF-I and IGF-II in peri-implantation and postimplantation conceptuses from normal and streptozotocin-treated diabetic mice. Animals were sacrificed on gestational days 5, 6, 7, 8, and 9. The entire uterine horn was prepared for hybridization with antisense and sense α-35S-dATP labeled oligonucleotide probes for IGF-I, IGF-II, and mouse β-actin. IGF-I transcript was apparent only in myometrium at 6 days of gestation in normal and diabetic mice. IGF-II transcripts were restricted to trophoectoderm cells within the implantation chamber on day 5. Following implantation, IGF-II transcripts were found in trophoectodermal derivatives, primitive endoderm, mesoderm, heart, walls of the foregut, and mesenchyme in normal and diabetic postimplantation conceptuses. There were no apparent differences between normal and diabetic samples in the distribution and abundance of the IGF-II transcript from gestational days 7, 8, and 9. The embryos from the diabetic mother at day 6 were growth retarded and had a significant decrease in the expression of IGF-II. These results suggest that maternal hyperglycemia may retard development of the early implanting conceptus in a narrow window around day 6 through a mechanism involving decreased IGF-II expression. Fetuses from diabetic pregnancies that escape this critical period appear to develop and express IGF-II in an equivalent manner to those of the control group. © 1994 Wiley-Liss, Inc.
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  • 179
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    Molecular Reproduction and Development 37 (1994), S. 398-406 
    ISSN: 1040-452X
    Keywords: Preimplantation embryo ; Insulin ; IGFs ; Protein synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Previously constructed protein databases for two stages of preimplantation mouse embryogenesis, the compacted eight-cell stage and the fully expanded blastocyst stage, have been used to analyze the effects of insulin, IGF-I, and IGF-II on protein synthesis in these developmental stages. Proteins were labeled by placing, for 2 hr, synchronous cohorts of 35-50 embryos into human tubal fluid (HTF) medium containing L-[35S]-methionine (1 mCi/ml) in the presence or absence of one of the growth factors. The embryos were then washed with medium and lysed. Samples were processed for 2-D gel analysis. For each embryonic stage and each growth factor, four or five experimental replicates were done and the gel images were compared using the PDQUEST system. Using the computer-assisted analysis, we were able to identify proteins that showed a statistically significant (P 〈 0.05) change in synthesis. At the eight-cell stage of development insulin caused increased synthesis of two proteins and decreased synthesis in three proteins. Insulin-treated blastocyst stage embryos exhibited an increased synthesis in eight proteins and decreased synthesis for one protein. The effect of IGF-I at the eight-cell stage of development was mostly inhibitory; the synthesis of only one protein increased and the synthesis of five proteins showed a decrease. Similar results were obtained with blastocyst stage embryos; four proteins demonstrated an increase in synthesis while 14 proteins showed a decrease. Eight-cell stage embryos incubated with IGF-II had seven proteins with a decreased synthesis, although in blastocyst stage embryos, nine proteins showed increased synthesis. However, seven IGF response proteins were found to be proteins that showed significant changes in isotope incorporation during the eight-cell to blastocyst stage of development (Shi et al., 1993). In all, 54 proteins were affected, and these were unique; thus, protein synthesis in preimplantation mouse embryos is influenced by insulin and the IGFs, and further, each growth factor affects specific proteins. © 1994 Wiley-Liss, Inc.
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  • 180
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    Molecular Reproduction and Development 37 (1994), S. 425-435 
    ISSN: 1040-452X
    Keywords: Seminal fluid proteins ; Isoelectric focusing ; Molecular heterogeneity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (Mr) between 6 and 100 kDa. Many isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a Mr of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its Mr to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their Mr nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content. © 1994 Wiley-Liss, Inc.
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  • 181
    ISSN: 1040-452X
    Keywords: In vitro fertilization ; Fertilization-specific antibody ; Immunohistochemistry ; Mouse-hamster-human cross-reacting antibodies ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Several intrasplenic immunizations with batches of ∼15 or ∼30 zona-free, unfertilized mouse oocytes resulted in 200-300 hybrids, respectively, among which about 20 positive clones were selected from each fusion between splenic plasma cells and SP2/0 myeloma cells. When nonimmunized splenic plasma cells were used, only one antibody, showing weak immunoreaction, was obtained from ∼370 hybrids collected from 2 fusions. From one immunization with a total of 12 zona-free, unfertilized mouse oocytes, 15 positive clones were selected for further study. Eleven of these 15 antibodies reacted with antigens only in unfertilized oocytes but not in fertilized, pronuclear stage oocytes. Three antibodies, which recognized antigens in paraffin-embedded oocyte sections, did not label growing ovarian oocytes, indicating that the antibodies were specific to ovulated, unfertilized oocytes. These antibodies did not detect any antigen epitopes in the panel of tissues examined. The molecular weight of one antigen, corresponding to a IgM antibody that is present both in ooplasma and zona pellucida, was ∼116 kDa. Cross-reactivity to blots of unfertilized zona-free hamster oocytes was demonstrated by 6 antibodies and to unfertilized human oocytes by 7 antibodies. Three antibodies cross-reacted with both hamster and human oocytes. The study indicates that the intrasplenic immunization is an appropriate means of raising antibodies against unfertilized, zona-free mouse oocytes and that the method applied offers an easy way to select antibodies against human oocytes for functional studies. © 1994 Wiley-Liss, Inc.
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  • 182
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    Molecular Reproduction and Development 37 (1994), S. 457-461 
    ISSN: 1040-452X
    Keywords: Canine sperm ; Pyospermia ; Ultrastructure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The red wolf (Canis rufus) is an endangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the genetic potential of the stock. Electron microscopic observations were made in semen specimens obtained by electroejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutrophils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive pyospermia and spermophagy but this finding was not correlated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen quality. © 1994 Wiley-Liss, Inc.
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  • 183
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    Molecular Reproduction and Development 37 (1994), S. 477-477 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 184
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    Molecular Reproduction and Development 37 (1994), S. 477-477 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 185
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    Molecular Reproduction and Development 37 (1994), S. 467-472 
    ISSN: 1040-452X
    Keywords: Oocyte maturation ; Germinal vesicle breakdown ; Polar body ; LH/FSH ; Macaque ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The specific aim of this study was to determine the effects of gonadotropins in vitro upon the incidence of and precise time interval to germinal vesicle breakdown (GVB) and extrusion of the first polar body (PB1) in oocytes from nonstimulated rhesus monkeys. Cumulus-enclod germinal vesicle (GV) stage oocytes from 10 normal, cycling rhesus monkeys in the follicular phase of the menstrual cycle were cultured with either: (1) 1.0 μg/ml human follicle-stimulating hormone (hFSH), (2) 10 μg/ml human luteinizing hormone (hLH), (3) 1.0 μg/ml hFSH and 10 μg/ml hLH, or (4) no gonadotropins (controls). Oocytes (n = 234) were examined at 3-hr intervals from 0 to 21 hr and at 4-hr intervals from 24 to 52 hr for GVB and PB1. Neither the incidence of GVB (hFSH: 63.5%; hLH: 56.1%; both gonadotropins: 63.1%; no gonadotropins: 53.6%) nor extrusion of PB1 (hFSH: 41.3%; hLH: 36.4%; both gonadotropins: 36.9%; no gonadotropins; 31.9%) differed (P 〉 0.05) among treatments. The time to GVB was accelerated (P 〈 0.05) by gonadotropins (hFSH: 10.8 ± 1.7 hr; hLH: 10.1 ± 1.8 hr; both gonadotropins: 8.8 ± 1.1 hr) when compared to controls (17.4 ± 2.0 hr). However, the time interval to extrusion of PB1 did not differ (P 〉 0.05) among treatments (hFSH: 32.3 ± 1.2 hr; hLH: 35.1 ± 1.4 hr; both gonadotropins: 35.2 ± 1.3 hr; no gonadotropins: 34.1 ± 1.2 hr). The mean interval to extrusion of PB1 was 34.1 ± 0.6 hr. In conclusion, GVB and PB1 extrusions appear to be, in part, independently regulated events in macaque oocytes matured in vitro since the timing of PB1 extrusion is not tightly coupled with the onset of GVB. Although the developmental potential of oocytes may be enhanced by gonadotropins, alternative approaches must be developed to improve the poor competence of oocytes from nonstimulated monkeys to mature in vitro. © 1994 Wiley-Liss, Inc.
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  • 186
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    Molecular Reproduction and Development 38 (1994), S. 48-53 
    ISSN: 1040-452X
    Keywords: Fertilization ; Eggshell ; Electrophoresis ; Dicentrarchus ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Fish eggs are surrounded by a resistant acellular coat commonly called the chorion or zona radiata. This study characterizes the eggshell proteinaceous content of unfertilized eggs of the sea bass Dicentrarchus labrax, with a view to the prepration of immunogens. Solubilization of the purified eggshells was achieved in 8 M urea followed by one- and two-dimensional gel electrophoresis. Glycoproteins were detected using concanavalin-A in one and two-dimensional gels, and the principal glycoproteins had a molecular weight of 47 kDa and 170 kDa. Partial purification of a few polypeptides in the 45 kDa to 55 kDa range was achieved by gel filtration chromatography. Although whole eggshells were relatively insoluble even in 8 M urea, partial purification of these polypeptides enable them to dissolve completely in solutions at low ionic strength. © 1994 Wiley-Liss, Inc.
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  • 187
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    Molecular Reproduction and Development 38 (1994), S. 43-47 
    ISSN: 1040-452X
    Keywords: Spermatozoa ; Sperm maturation ; Sperm association ; Epididymis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Recently, a new head-to-head sperm association was described in the rat during epididymal transit. This association was called a rosette and a filamentous and PAS-positive material was also described joining the sperm heads. The begining of rosette formation in the epididymis and the linking material between heads have remained unclear. Epididymides of adult rats were fixed by vascular perfussion and thin sections of the principal regions were studied by transmission electron microscopy (TEM). The first evidence of rosette formation was observed in the distal corpus. Rosettes were isolated from the distal corpus and processed for immunogold and immunofluorescence microscopy to detect an epididymal glycoprotein called DE. This glycoprotein is secreted by the corpus epididymis and appears to be involved in sperm maturation. Colloidal gold marks and fluorescence were observed in the linking material between the sperm heads. The results presented here show that rosettes begin to appear following the sites of DE secretion and permit us to postulate that DE is involved in rosette formation and constitutes another example of gamete-epididymal interaction. © 1994 Wiley-Liss, Inc.
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  • 188
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    Molecular Reproduction and Development 38 (1994), S. 69-76 
    ISSN: 1040-452X
    Keywords: Hyaluronic acid binding protein ; Sperm motility ; Phosphorylation ; Epididymal maturation ; Fertilization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Hyaluronic acid, a major component of the extracellular matrix, plays an important role in the regulation of different cellular processes, e.g., locomotion, cell-cell interaction during morphogenesis, and differentiation. Distribution of hyaluronic acid with respect to the role of sperm hyaluronidase in sperm penetration and gamete interaction is well established. In order to elucidate this mechanism, in our current study we have identified and demonstrated, for the first time, the presence of a 68-kDa cell surface hyaluronic acid binding glycoprotein (HABP) in spermatozoa of different species (rat, mice, bull, and human) by immunoblot analysis and indirect immunofluorescence using the polyclonal antibodies raised against purified HABP. Furthermore, we were able to demonstrate a differential distribution of 68-kDa HA binding protein on the sperm head, midpiece, and tail of different species. To identify its role in sperm function, we observed its declining pattern during epididymal maturation and also the inhibition of sperm-oolemmal adherence by pretreatment of the sperms with anti-HABP antibodies. We have further observed its in vivo phosphorylation in motile spermatozoa. All our data clearly indicate that sperm hyaluronan binding protein may have a specific role in sperm maturation, motility, and fertilization processes. © 1994 Wiley-Liss, Inc.
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  • 189
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    Molecular Reproduction and Development 38 (1994), S. 30-35 
    ISSN: 1040-452X
    Keywords: Rat embryo ; Transcription ; Protein synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Expression of the embryonic genome has been examined during preimplantation rat development. Proteins synthesized at different stages of embryogenesis were labelled with [35S]methionine and then separated by one-dimensional gel electrophoresis. A major transformation in the pattern of protein synthesis has been observed between the two- and the four-cell stages of embryonic development. Also the culture of embryos with an inhibitor of transcription (α-amanitin) has shown that the first α-amanitin-sensitive events take place during the late two-cell stage. However, inhibition of transcription does not arrest the embryo development up to the four-cell stage. Taken together, the results indicate that in rats the initiation of embryonic gene activation occurs at the late two-cell stage. However, the first two cleavage divisions can occur in the absence of transcription. © 1994 Wiley-Liss, Inc.
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  • 190
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    Molecular Reproduction and Development 38 (1994), S. 36-42 
    ISSN: 1040-452X
    Keywords: Human blastocyst formation ; Frozen sperm ; Abnormal sperm ; Human I.V.F. ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In human in vitro fertilization (I.V.F.), it was first assumed that all the embryos obtained had the same developmental potential whatever the quality of sperm. However, this has not been confirmed. We have used the coculture technique and determined the blastocyst formation rate in three groups of patients: group 1: patients with normal sperm count (〉20 × 106/ml), motility (〉30%), and morphology (〉50%); group 2: patients treated by I.V.F. with frozen donor sperm; group 3: patients with severely impaired sperm quality (〈3 × 106 forward motile and morphologically normal spermatozoa per ml). In group 1, we found a strong correlation between cleavage rate and blastocyst formation rate (P 〈 0.0001) with a blastocyst formation rate comprised between 40% and 50%. This was not true for the two other groups for which the overall number of blastocysts obtained and the number of patients having at least one blastocyst were severely reduced (P 〈 0.0001). These data are discussed in terms of DNA quality, timing of formation of the pronuclei, and delays in cell cycles at the time of genomic activation. These observations lead to a new approach to the study of fertilizing ability of poor quality sperm. It may help in the decision as to whether couples treated for male infertility should be excluded from I.V.F. protocols. © 1994 Wiley-Liss, Inc.
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  • 191
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    Molecular Reproduction and Development 38 (1994), S. 54-60 
    ISSN: 1040-452X
    Keywords: Mouse spermatozoa ; Subzonal insemination ; Calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of calcium concentration in culture medium on the fertilization of subzonally microinseminated mouse oocytes was examined. Oocytes were injected with a single spermatozoon so that the sperm head was forced to adhere onto the ooplasmic membrane with a micromanipulation technique. For the inseminations, epididymal spermatozoa preincubated in culture medium and those treated with ionophore A23187 were used. Inseminated oocytes were cultured using media with three different calcium concentrations of 1.71, 3.42, and 5.13 mM; 40.0%, 71.6%, and 47.9% of oocytes microinjected with preincubated sperm were fertilized after incubation with those media, respectively. When the oocytes inseminated with ionophore-treated sperm were incubated in media containing 1.71 and 3.42 mM calcium, their fertilization rates were 58.2% and 87.5%. Thus fertility of subzonally microinseminated oocytes was obviously enhanced when cultured in medium with 3.42 mM of calcium, irrespective of being inseminated with preincubated sperm (P 〈 0.01) or with ionophore-treated sperm (P 〈 0.005). Some of the microinseminations with preincubated sperm were performed without sperm adhered to the oolemma. In these cases, the incidence of fertilization was not improved by incubating the inseminated oocytes in medium containing 3.42 mM calcium (32.6%) as compared to those incubated in medium with 1.71 mM calcium (28.3%). These results suggest that the concentration of extracellular calcium exerts an important effect on the progress of fertilization events subsequent to sperm adherence onto the ooplasmic membrane. Almost 80% of the zygotes fertilized via incubation in medium with 3.42 mM of calcium developed into blastocysts after culturing in vitro. © 1994 Wiley-Liss, Inc.
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  • 192
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    Molecular Reproduction and Development 38 (1994), S. 61-68 
    ISSN: 1040-452X
    Keywords: Nuclear transfer ; Bovine ; Activation ; Oocyte ; Embryo ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Successful bovine nuclear transfer (NT) embyro production requires proper oocyte activation and transfer of a nucleus into this oocyte. However, the temporal relationship between these two events is unclear. The current study examined whether activation of the oocyte prior to fusion would induce nuclear swelling while also affecting development to morula and blastocyst stage and finally development to offspring. Aged oocytes can be activated by a number of techniques including exposure to room temperature. In this study oocyte activation was induced through three different means: reduced temperature culture alone, reduced temperature culture and calcium ionophore, and naturally, through the fertilization process. Electrofusion was carried out after the activation stimulus. When used in the NT procedure, activation of oocytes prior to fusion resulted in NT embryos that under went nuclear swelling and had a high developmental rate to morula and blastocyst stages. Also, these NT embryos developed to normal offspring when transferred to recipient animals. The addition of a calcium ionophore treatment to the reduced temperature culture was not beneficial and resulted in less nuclear swelling. The use of enucleated fertilized oocytes as recipient cytoplasm for the new nucleus resulted in NT embryos developing to morula and blastocyst stages at the same rate as room temperature activated NT embryos. Therefore, improved embryo development can be obtained from NT embryos if the aged recipient oocyte is activated prior to the time of fusion. Also, offspring were obtained from these pre-activated NT embryos. © 1994 Wiley-Liss, Inc.
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  • 193
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    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 91-93 
    ISSN: 1040-452X
    Keywords: Zona pellucida ; Egg activation ; Mouse ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A rapid, nonradioactive method to monitor the ZP2 to ZP2f conversion in the zona pellucida of single mouse eggs has been developed. This assay is based on the chemiluminescent detection of biotinylated ZP2 and ZP2f following electrophoresis under reducing conditions and electrophoretic transfer to Immobilon P. This method is about 10 times faster and detects similar extents of ZP2 to ZP2f conversion following A23187-induced egg activation, when compared to the commonly used radioiodination procedures. © 1994 Wiley-Liss, Inc.
    Additional Material: 2 Ill.
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  • 194
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 94-106 
    ISSN: 1040-452X
    Keywords: Epididymal spermatozoa ; Motility patterns ; VCL ; VSL ; VAP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A computer-aided semen analysis system was used for the objective assessment of hamster spermatozoa during epididymal maturation. The caput epididymal spermatozoa were extremely sluggish, achieved very little progression, and the three velocity parameters, namely curvilinear velocity (VCL), progressive velocity (VSL), and path velocity (VAP), were low. These spermatozoa during progressive movement alternated between the linear shape and “U” shape or attained an “S” shape prior to changing to the “U”; shape. The corpus epididymal spermatozoa were faster, displayed greater VSL, VAP, and VCL compared to caput epididymal spermatozoa, and, during forward motility, attained “U,” “C,”; and (or) “?” shape as in the wriggling motility pattern. The proximal cauda epididymal spermatozoa were actively motile and VSL, VAP, and VCL in these spermatozoa were more than 10 times greater compared to the caput epididymal spermatozoa. The proximal cauda epididymal spermatozoa predominantly moved in circles and with time became slower and more circular in their trajectories and exhibited a reduction in LIN (linearity). The distal cauda epididymal spermatozoa were very similar to the proximal cauda epididymal spermatozoa with respect to their fast motility (VSL, VAP, and VCL are similar) and beat cross frequency (BCF), but showed larger values for STR (straightness) and LIN and moved along curved trajectories. The amplitude of lateral head displacement (ALH) was also considerably lower in the distal cauda epididymal spermatozoa compared to the proximal cauda epididymal spermatozoa. Thus, this study provides for the first time data related to seven motility parameters for caput and corpus epididymal spermatozoa of hamster. It also provides additional data with respect to VCL, LIN, BCF, and ALH for proximal and distal cauda epididymal spermatozoa of hamster. © 1994 Wiley-Liss, Inc.
    Additional Material: 11 Ill.
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  • 195
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    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 119-130 
    ISSN: 1040-452X
    Keywords: Mouse embryos ; Nuclear antigens ; Cell proliferation ; sn-RNP ; Transcriptional activity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have systematically analyzed by indirect immunofluorescence the subcellular distribution of nuclear antigens in relation to developmental stages of maturing mouse oocytes and developing embryos. Antigens were of two types: (1) a protein whose nuclear localization in interphase somatic cells depends on their proliferative state protein recognized by a monoclonal antibody 43B1N, and (2) snRNP polypeptides recognized by autoimmune sera of anti-Sm and anti-RNP type. The protein recognized by 43B1N was present in the germinal vesicle of oocytes from antral follicles, but absent from the nuclei during the first hours of embryonic life up to the middle to late 2-cell stage. Starting from this stage, it was always found in nuclei of interphase blastomeres, where its “speckles”; co-localized with the speckles containing high concentrations of snRNP polypeptides. SnRNP polypeptides recognized by anti-Sm and anti-RNP sera were in turn found in nuclei of all developmental stages. When embryos were treated with aphidicolin or cytochalasin D to arrest cell division, the 43B1N reacting protein was again localized in the pronuclei at 42 hr post-hCG, i.e., slightly later than the onset of transcriptional activity. These results suggest a progressive building up of nuclei during embryonic development, which could influence gene expression. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 196
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    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994) 
    ISSN: 1040-452X
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 197
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    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 107-117 
    ISSN: 1040-452X
    Keywords: Masked mRNA ; mRNPs ; mRNA-binding proteins ; Translational repression ; mRNA stability ; mRNA storage in germ cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Messenger RNA can be stored in the cytoplasm of higher Eukaryotes in the form of masked messenger ribonucleoprotein particles (masked mRNPs, or informosomes). The typical example is the storage of mRNPs in germ cells (oocytes and spermatocytes). The masked mRNPs are inactive in translation, stable, i.e., protected against degradation, and unavailable for poly(A) tail processing, such as cytoplasmic polyadenylation and deadenylation. The major nonspecific mRNA-binding protein forming mRNPs and belonging to a special p50 family of basic, glycine-rich, phosphorylatable proteins seems to be necessary, but not sufficient for the masking. In some cases, mRNA-specific repressor proteins bound to the 5′-untranslated regions (5′-UTR) of mRNAs may be involved. Interactions of the 3′-untranslated regions (3′-UTR) with sequence-specific proteins seem to be of decisive importance for the masking of mRNPs. The hypothesis is proposed that the masking is achieved through a 3′-UTR-induced conformational rearrangement of mRNP; closing into a circle and condensation of mRNP are considered plausible. © 1994 Wiley-Liss, Inc.
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  • 198
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    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 142-147 
    ISSN: 1040-452X
    Keywords: Micromanipulation ; Zona pellucida ; DNA repair ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The treatment of male factor-related infertility has been approached with the advent of several methods for microsurgical fertilization, such as the partial dissection of the zona pellucida (PZD) and the injection of sperm into the perivitelline space (PVSI) of oocytes. These techniques are designed to increase sperm-oolemma interaction by circumventing passage of the sperm through the zona pellucida. The present study was performed to evaluate the influence of PZD and PVSI on the in vitro development of mouse embryos by assessing the rate of sister chromatid exchange (SCE). SCE is considered to be a sensitive indirect indicator of DNA lesions due to various conditions. Oocytes were cultured in vitro after PZD or PVSI and then examined for SCE. There was no significant difference in SCE between control and treatment groups of embryos and the values were similar to those reported by Saito et al. (Fertil Steril 41:460-464, 1984). The rate of SCE was low during the first two mitotic cycles, then increased from cycle two to three before declining slightly between the 3rd and 4th cycles of cell division. These data demonstrate that the direct interaction of sperm and oocyte by PZD or PVSI did not have an adverse effect on the development of mouse embryos as assessed by the rate of SCE. © 1994 Wiley-Liss, Inc.
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  • 199
    ISSN: 1040-452X
    Keywords: Oviductal glycoprotein ; Gametes ; Immunofluorescence ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The objective of this study was to determine if human oviduct specific glycoprotein (huOGP) would associate with hamster ovarian oocytes and human sperm during in vitro incubation. The huOGP used in these studies was partially purified from human hydrosalpinx fluid. Hamster ovarian oocytes and human sperm samples were incubated in culture medium with and without huOGP. Association of huOGP was assessed by indirect immunofluorescence assay using a polyclonal antibody prepared against huOGP. Intense fluorescence of the zona pellucida, and bright but uneven fluorescence of the perivitelline space, were observed in hamster ovarian oocytes following incubation in the presence of huOGP. A similar but more uniform pattern of fluorescence was observed when hamster oviductal oocytes (positive controls) were incubated in culture medium alone. Fluorescence was absent when oocytes were assayed with preimmune serum. The association of huOGP with the zona pellucida and perivitelline space appeared to be specific since thyroglobulin, a large molecular weight glycoprotein, and human serum albumin, the major protein in oviduct fluid, did not associate with the hamster oocytes nor inhibit huOGP association when included in the culture medium. Fluorescence was absent when human sperm incubated with huOGP were assayed with antiserum to huOGP. However, human sperm fluoresced when incubated with a uterine glycoprotein, CUPED, which had previously been shown to bind to cat sperm during in vitro incubation. Sperm also fluoresced brightly when human sperm antibody was used as a positive control. Solubilization of sperm membrane proteins postincubation and analysis of these proteins by 1-D SDS-PAGE followed by immunoblotting also failed to show an association of huOGP with human sperm. Electron microscopy of sperm both pre- and postsolubilization confirmed that the sperm membranes were removed by this process. In conclusion, the association of huOGP with hamster oocytes in vitro suggests that huOGP may associate with human oocytes in vivo, whereas that may not be true for human sperm in vivo. The association of huOGP with oocytes may serve to facilitate the process of fertilization and early embryonic development within the oviduct. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
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  • 200
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    New York, NY [u.a.] : Wiley-Blackwell
    Molecular Reproduction and Development 38 (1994), S. 148-152 
    ISSN: 1040-452X
    Keywords: Leukemia inhibitory factor ; Rabbit ; Endometrium ; Blastocyst implantation ; Pregnancy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Leukemia inhibitory factor (LIF) has been shown to play an important role in the implantation of mouse blastocysts. The present study was designed to document the appearance of LIF in the rabbit uterus during early pregnancy and to determine whether changes just prior to implantation, similar to those in mice, occurred. LIF was localized in endometrial epithelium, myometrium, and endometrial glands. A low level of LIF was detected in the uterus of nonestrous and estrous females. LIF expression reached its highest level on day 5 of pregnancy and declined on days 6 and 7. By day 13 of pregnancy, little endometrial LIF was apparent. The expression of LIF on day 5 of pseudopregnancy was similar to that on day 5 of pregnancy. LIF expression was much higher at implantation sites than that at nonimplantation areas on day 7 of pregnancy. It is concluded that LIF may be important for the implantation of rabbit blastocysts. © 1994 Wiley-Liss, Inc.
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