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  • Wiley-Blackwell  (13,694)
  • 1990-1994  (13,694)
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  • 1993  (13,694)
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  • 1990-1994  (13,694)
  • 1970-1974
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 2
    ISSN: 0886-1544
    Keywords: human fibroblast tropomyosins ; normal and transformed cells ; actin-binding protein ; cDNA cloning ; expression of tropomyosin isoforms ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A tropomyosin-specific oligonucleotide probe (REN29) designed to hybridize to all known human tropomyosin isoforms was used to study tropomyosin mRNA levels in normal and transformed human cells. At least four different sizes of RNAs were detected in normal human fibroblast KD cells by Northern blot analysis. The major bands of 1.1 kb RNA for hTM1 and 3.0 kb RNA for hTM4 were decreased substantially in various transformed cell lines. One of the minor RNA bands (2.0 kb for hTM2 and hTM3) appeared to be absent in a human pancreatic carcinoma cell line. The level of the other minor RNA band (2.5 kb for hTM5) was found to be unchanged or slightly decreased in transformed cells. This differential expression of tropomyosin isoforms at the RNA level was not totally in agreement with the difference in the protein amounts found in normal and transformed cells, suggesting that translational control may also play an important role in the expression of some tropomyosin isoforms. The REN29 probe was further used to screen γgt10 and γgt11 cDNA libraries, which were constructed from poly(A)+ RNAs of human fibroblast cell lines HuT-14 and WI-38, respectively. In addition to cDNA clones encoding known isoforms, we obtained three classes of new cDNA clones that encode two low Mr isoforms (hTM5a and hTM5b), and a high Mr isoform (hTMsmα). Sequence comparison revealed that hTM5a and hTM5b are alternatively spliced products derived from the same gene that encodes hTM2 and hTM3. Northern blot analysis and amino acid sequence comparison suggested that the hTMsmα represents a smooth muscle tropomyosin which is also expressed in human fibroblasts. The exon specific for, and common to, hTM5a and hTM5b was found to be highly expressed in small intestine. However, there was no detectable expression of this exon in stomach and skeletal muscle. The difference in tissue-specific expression suggests that different isoforms may perform distinct functions in different tissues. © 1993 Wiley-Liss, Inc.
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  • 3
    ISSN: 0886-1544
    Keywords: MTOC ; dendrites ; neurite extension ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Centrosomes are unique cytoplasmic structures which serve as microtubule organizing centers (MTOC). In most animal cells centrosomes consist of one or more pair of centrioles surrounded by electron dense amorphous pericentriolar material (PCM) responsible for nucleation of microtubules. In the present study we analyzed the pattern of induction and localization of proteins of the PCM at different stages of neuronal development in cell cultures prepared from the embryonic hippocampus. For this purpose we used a human polyclonal antibody that recognizes two proteins of the PCM (100 kd and 60 kd, respectively). The results indicate that in mature neurons, pericentriolar immunoreactive material is preferentially localized in dendritic processes, and that throughout the course of neurite development and differentiation it is systematically excluded from the neuron's axon. Western blot analysis showed that during neuronal development in situ, there is an increase in he immunoreactivity for both proteins recognized by this antibody. In contrast, in hippocampal pyramidal neurons that develop in culture, there is an increase in the 60 kd polypeptide, while the 100 kd one is not detected after 7 days in vitro. © 1993 Wiley-Liss, Inc.
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  • 4
    ISSN: 0886-1544
    Keywords: actin ; actin-binding protein ; capping protein ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Actin filaments undergo dramatic changes in their organization during myofibrilloenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z-discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to “cap” each actin filament of the sacromere. Double inmmunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, α-actinin, titin, and myosin during myofibrilloenesis. Of particular interest was the assembly of CapZ at nascent Z-discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non-striated actin filaments called stress fiber-like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z-discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I-bands, CapZ was distributed in a periodic pattern characteristic of mature Z-discs. The finding that CapZ was assembled at nascent Z-discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I-band formation in skeletal muscle cells. The assembly of CapZ at nascent Z-disc structures also was observed relative to the assembly of sarcomeric α-actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z-discs. The distribution of CapZ and sarcomeric α-actinin in young myotubes was not coincident; in older myotubes, both CapZ and α-actinin were co-localized at Z-discs. In cardiac myocytes, CapZ was detected at Z-discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co-localized with A-CAM and vinculin at cell-cell junctions formed by the myocytes. © 1993 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 25 (1993), S. 358-368 
    ISSN: 0886-1544
    Keywords: smooth muscle ; smooth muscle myosin ; nonmuscle myosin ; myosin isoforms ; cellular myosin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In smooth muscle tissue, two or three isoforms of myosin heavy chain (MHC) have been reported (SM1, SM2, and/or NM). In mouse uterus tissue, four bands in the region of the MHC's can be resolved on high resolution SDS polyacrylamide gels. Western blots using smooth muscle (SM) MHC-specific and nonmuscle (NM) MHC-specific polyclonal antibodies show the upper two bands in the MHC region are SM isoforms, whereas the lower two bands are NM isoforms. One-dimensional peptide maps of these four bands show each to have a unique pattern of polypeptide fragments following α-chymotrypsin digestion. Developmental expression of myosin heavy chains (MHC) in mouse uterus, aorta, bladder, and stomach (6 ages, 10-150 days) was determined using tissue homogenates. In the uterus, both SM MHC's show an increase in relative content with increasing age, whereas the NM MHC's show a decrease. The mouse aorta shows a significant increase in the SM MHC's and a significant decrease in the NM MHC from day 10 to day 30, which is similar to data reported for the rat aorta. Whereas both the bladder and stomach contain relatively small amounts of NM MHC's (∼ 10% or less), these quantities do show decreases with development. The SM1:SM2 ratio for the uterus remains high (3.4 at 150 days) through development; the aorta, bladder, and stomach also start out high, but tend toward 1.0 in the 150-day animals. The presence of four MHC isoforms in the uterus with unique developmental regulation of expression is consistent with hypotheses of unique functional roles for these isoforms. © 1993 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 7
    ISSN: 0886-1544
    Keywords: polymorphonuclear leukocytes ; quantitative fluorescence microscopy ; lysed cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We compared, on lysed polymorphonuclear leukocytes (PMNs), the spatial distributions of sites that nucleate actin polymerization with the spatial distribution of endogenous F-actin. Sites nucleating polymerization of exogenous actin were detected by incubating lysed cells with rhodamine-labeled G-actin under polymerizing conditions. Endogenous F-actin was stabilized and stained by lysis of cells into fluorescein-labeled (FITC) phalloidin. We found the distributions of rhodamine and fluorescein intensities in a given cell, resting or stimulated with chemoattractant, to be similar. Thus, after lysis the number of sites able to nucleate actin polymerization is proportional to the local F-actin concentration.Quantitative fluorescence microscopic analysis also demonstrated that (1) if cells were stimulated with chemoattractant shortly before lysis, the total fluorescence per cell of both fluorophors went up; (2) if peptide was diluted shortly before lysis, the endogenous F-actin in the lamellae was dramatically reduced, but nucleation sites persisted, giving a high rhodamine to fluorescein ratio; and (3) there was a small increase in the ratio of rhodamine (exogenously grown actin) to fluorescein (endogenous F-actin) in a region near the lamellar/endoplasm border, centripetal to regions of the highest concentration of endogenous F-actin.The rhodamine signal appeared to be due to in situ actin polymerization probably nucleated by existing free barbed ends, since (1) the rhodamine signal increased linearly with time with no detectable lag if the actin concentration was above that of the critical concentration of the barbed end; (2) the rhodamine signal was dramatically reduced if lysates were incubated with gelsolin-actin complex (which stably caps barbed ends), then washed before the rhodamine G-actin was added; and (3) the number of nucleation sites at the time of lysis is similar to the number of the barbed ends of actin filaments determined by the kinetics of depolymerization [Cano et al., 1991].The fact that the distribution of exogenous actin polymerization paralleled the endogenous F-actin suggests that the number of free barbed ends per F-actin is roughly constant. If all filament ends were free, or if a constant fraction of the filaments ends were free, these data would suggest that the mean filament length is roughly constant throughout the cell. © 1993 Wiley-Liss, Inc.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 25 (1993), S. 309-316 
    ISSN: 0886-1544
    Keywords: F-actin ; polymerization ; depolymerization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cell locomotion depends on polymerization and depolymerization of filamentous actin. Net polymerization at the cell front occurs fast enough to fill the extending lamellipod, and since total F-actin is essentially constant over time, depolymerization must equal polymerization. Indeed, the fastest moving cell types have the highest rates of depolymerization. Accounting for the high rate of depolymerization raises several problems. One is that net depolymerization requires the concentration of G-actin to be low (below the critical concentration), but rapid polymerization (occurring 〈1 μm away) requires the concentration of G-actin to be high (well above the critical concentration). This may be accomplished by spatial compartmentalization of factors that favor polymerization or depolymerization, and/or by proteins that bind G-actin and prevent spontaneous polymerization while allowing barbed-end elongation. A second problem is that depolymerization proceeds faster than would seem possible from studies of F-actin in vitro (as calculated from number and lenghts of filaments present and in vitro rate constants). Rapid depolymerization may be accomplished by filament cutters or by cytoplasmic components (as yet undiscovered) that increase the rate of depolymerization. © 1993 Wiley-Liss, Inc.
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  • 10
    ISSN: 0886-1544
    Keywords: fluorescent analogue cytochemistry ; cytoskeletal transport ; photobleach technology ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have examined the effects of various means of photobleaching on the recovery of fluorescene, movement, and morphology of the microtubules in the neurites of rhodamine-tubulin-injected PC12 cells. We find that, depending on power of and time of exposure to the bleaching beam, we can generate at least three different patterns of fluorescence recovery in regenerating PC12 neurites. If bleaching is performed with a relatively low-power beam for an extended period, fluorescence in polymer recovers very little after 1 hours. Under these conditions, however, tubulin immunostaining is seen extending through the bleach zone, and microtubules are present through the bleached zone in thin section electron micrographs. If bleaching is performed with a high-power laster, for 0.5-5 seconds, fluorescence recovery also is quite slow, but electron microscopic observations reveal that no microtubules extend through the bleached region of the neurite, and the uranyl acetate-stained cytoplasm appears more electron lucent than in the unbleached neurite. Finally, if bleaching is performed by very brief exposure to a high-intensity laser beam, resulting in an incomplete reduction of fluorescence intensity through the bleach zone, fluorescence recovery occurs within 20-30 minutes, and immunostained microtubules appear intact through the bleach zone; electron microscopy confirms that microtubules extended through the bleached zone of such neurites. In all three cases, movement of the bleach zone is observed in approximately half of the experimental neurites. These results indicate that highly variable microtubule behaviors can be obtained with photobleach technology, presumably due to different levels and pathways of photodamage generated by different bleach protocols. Nevertheless, it is clear that both turnover and movement of microtubules occur in FC12 neurites, and both are likely to be involved in neurite maintenance and growth. © 1993 Wiley-Liss, Inc.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 12
    ISSN: 0886-1544
    Keywords: myosin heavy chain ; cytokinesis ; dephosphorylation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Myosin heavy chain (MHC) phosphorylation was examined throughout the period of first cleavage in developing sea urchin embryos. MHC was found to be phosphorylated in these cells and, furthermore, the relative state of myosin phosphorylation was found to decrease as cells progressed through cytokinesis. Following the completion of cytokinesis, the relative state of MHC phosphorylation returned to levels observed in precytokinesis cells. The above results were obtained with myosin immunoprecipitated from whole cell lysates. In order to specifically examine the phosphorylation, state of MHC in the cleavage furrow, a protocol was developed for the isolation of intact contractile rings from dividing sea urchin embryos. MHC was immunoprecipitated from isolated contractile rings and the relative phosphorylation state of this MHC was compared with that of MHC isolated from whole cell lysates prepared at the same time. MHC from isolated contractile rings was found to be significantly less phosphorylated than total cellular MHC, suggesting a role for MHC dephosphorylation during cytokinesis. © 1993 Wiley-Liss, Inc.
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  • 13
    ISSN: 0886-1544
    Keywords: pancreas MAPs ; microtubule-affinity chromatography ; 67 kDa polypeptide ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have identified a 67 kDa heat-stable protein among the proteins which bind specifically to brain microtubules immobilized on a chromatographic support. Its relationship to tubulin and to the cytoskeleton using polyclonal antibodies has been studied. This 67 kDa protein is present in cytoskeleton and microtubule preparations from pancreas. This heat-stable microtubule-associated protein (MAP) copolymerized with phosphocellulose purified brain tubulin. The 67 kDa polypeptide was immunoreactive to antibodies against the 210 kDa MAP from HeLa cells; it also reacted with antibodies against an oligopeptide whose sequence corresponded to the second repeat of mouse brain tau. © 1993 Wiley-Liss, Inc.
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  • 14
    ISSN: 0886-1544
    Keywords: actin ; tubulin ; vimentin cytoskeleton ; cataract ; elasmobranch lens ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ultraviolet radiation in the near range (UVA) causes lens opacification and disrupts the actin cytoskeleton in rabbit and gray squirrel lenses. Changes were noted using transmission electron microscopy of tangential sections and rhodaminephalloidin fluorescence microscopy of epithelial whole mounts of irradiated and unirradiated lenses, and corresponded with gross cataract formation. Irradiated lenses lacked microfilament polygonal arrays at the inner surface of the apical plasma membrane (i.e., in the cell pole next to the lens fibers) in lens epithelia of both species; a condensed actin bundle was present instead. This bundle, and scattered small actin clumps in the cytoplasm, were identified by immunogold TEM, using a specific antibody and a secondary antibody conjugated with coloidal gold. Similar techniques showed breakdown of tubulin and vimentin, but after longer intervals than for the breakdown of actin. Generalized cytologic damage was also present in epithelial cells, but not in the underlying cortical lens fibers. Damage began to occur after 4 hr of irradiation and became more severe with increased exposure. Shielded controls remained clear, had normal cytology and polygonal arrays, and no clumping of actin filaments. © 1993 Wiley-Liss, Inc.
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  • 15
    ISSN: 0886-1544
    Keywords: endoplasmic reticulum ; carbocyanine dyes ; mitosis ; cell division ; membranous organelles ; confocal microscopy ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution and dynamics of the membranous organelles in two cell types were investigated during cell division. Live cells (either PtK2 or LLC-PK1) labeled with the vital dye 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)] were observed via serial optical sectioning with the laser-scanning confocal microscope. Z-series of labeled, dividing cells were collected every 1-2 minutes throughout mitosis, beginning at prophase and extending to the spreading of the daughter cells. Membrane distribution began to change from the onset of prophase in both cell types. When the mitotic spindle formed in prometaphase, fine tubular membranes, similar to those extending out to the edges of interphase cells aligned along the kinetochore spindle fibers. The lacy polygonal network typical of interphase cells persisted beneath the spindle, and a membrane network was also associated with the dorsal layer of the cell. As PtK2 cells reached metaphse, their spindles were nearly devoid of membrane staining, whereas the spindles of LLC-PK1 cells contained many tubular and small vesicular membranous structures. X-Z series of the LLC-PK1 metaphase spindle revealed a small cone of membranes that was separated from the rest of the cytoplasm by kinetochore MTs. In both cell types, as chromosome separation proceeded, the interzone remained nearly devoid of membranes until the onset of anaphase B. At this time the elongating interzonal microtubules were closely associated with the polygonal network of endoplasmic reticulum. Cytokinesis caused a compression, and then an exclusion of organelles from the midbody. Immunofluorescence staining with anti-tubulin antibodies suggested that spindle membranes were associated with microtubules throughout mitosis. In addition, taxol induced a dense and extensive collection of small vesicles to collect at the spindle poles of both cell types. Nocodazole treatment induced a distinct loss of organization of the membranous components of the spindles. Together these results suggest that microtubules organize the membrane distribution in mitotic cells, and that this organization may vary in different cell types depending on the quantity of microtubules within the spindle. © 1993 Wiley-Liss, Inc.
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  • 16
    ISSN: 0886-1544
    Keywords: fatty acid ; MHC ; MLC ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The fatty acid 12(S)-HETE may be a new second messenger capable of activating PKC. In tumor cells 12(S)-HETE stimulates cytoskeleton-dependent cellular responses such as adhesion and spreading. Analysis of 12(S)-HETE effects on B16a melanoma cell cytoskeleton revealed reversible rearrangement of microtubules, microfilaments, the actin-binding proteins, vinculin, myosin heavy (MHC) and light chains (MLC), as well as bundling of vimentin intermediate filaments. The alterations in microfilaments and intermediate filaments occurred very rapidly, i.e., 5 min after exposure of tumor cells to 12(S)-HETE. The 12(S)-HETE-induced cytoskeletal alterations were accompanied by centrifugal organelle-translocation. Interestingly, MLC exhibited clear association with the cytoplasmic organelles. Biochemical analysis of the 12(S)-HETE effect indicated a PKC-mediated reversible hyperphosphorylation of MLC, vimentin, and a 130 kD cytoskeletal-associated protein. Optimal effects were obtained after 5 min treatment with 12(S)-HETE at 0.1 μM concentration. 12(S)-HETE pretreatment induced tumor cell spreading on a fibronectin matrix which required the intactness of all three major cytoskeletal components. The spreading process was dependent upon the activity of PKC. Our data suggest that 12(S)-HETE is a physiological stimulant of PKC. Further, it induces rearrangement of the cytoskeleton of tumor cells in interphase resulting in the stimulation of cytoskeleton-dependent cell activity such as spreading. © 1993 Wiley-Liss, Inc.
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  • 17
    ISSN: 0886-1544
    Keywords: cell surface iodination ; intermediate filaments ; cell surface keratins ; keratins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Keratins are a subgroup of cytoskeletal intermediate filament proteins found in most epithelial cells. Some reports have suggested that keratins may be found on the cell surface as well as their well-accepted cytoskeletal location. A major part of the evidence in the interpretation of cell surface expression of keratins is cell surface radioiodination. Here we show that lactoperoxidase-catalyzed iodination of colonic and breast tissue culture cells results in radiolabeling of the keratins when cells are manipulated. No labeling of keratins is detected when cells are labeled directly on the tissue culture dish. A similar result was obtained when intact cells were biotinylated using water-soluble sulfo-NHS-biotin. Partitioning of the keratins to a soluble and an insoluble pool after “cell surface” 125I-labeling showed that both pools became iodinated. Indirect immunofluorescence showed that binding of a panel of anti-keratin antibodies to intact epithelial cells occurs only on the cells that are more adherent, which are the cells that require longer manipulation to remove from the tissue culture dish. Taken together, our results indicate that the reported expression of cell surface keratins in some cells likely reflects intracellular keratins. In addition, the method of epithelial cell handling can dramatically alter the leakiness of cell surface iodination techniques. © 1993 Wiley-Liss, Inc.
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  • 18
    ISSN: 0886-1544
    Keywords: nickel ; cadmium ; dynein ; motility ; flagella ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Bovine sperm, extracted with 0.1% Triton X-100, frozen at -20°C for 48-120 hours, and thawed, disintegrated by microtubule sliding when 1 mM MgATP was added. Microtubules and outer dense fibers (ODFs) were usually extruded in groups or “bundles.” A total of 44.5% of the cells extruded two distinct bundles, one from each side of the connecting piece, exhibiting opposite curvatures. Only one bundle was observed in 46.2% of the cells, and 9.2% showed no signs of sliding. Transmission electron microscopy (T.E.M.) showed one group consisting of the 4,5-6,7 elements, with the 9,1,2 elements on the other side of the axoneme making up the other bundle. T.E.M. revealed that when only one side of the axoneme had extruded elements, they were always from the 4,5-6,7 group. The remainder of the axoneme (8,9,1,2,3 and the central pair) was left relatively intact, suggesting a difference in the sliding response of the nine pairs of axonemal microtubules. These results indicate a predisposition for sliding between elements 7 and 8 over that between doublets 2 and 3, perhaps due to a disparity in activation thresholds. Also, both Ni2+ and Cd2+ appear to selectively block activation of 2-3 interdoublet sliding.Incubation with 0.25 mM Ni2+ prior to adding MgATP modified the percentages of sliding patterns: 8.6% demonstrated two-sided extrusion, 58.2% showed one-sided, and 33.2% had no extruded bundles. Again, when half the axoneme was missing, it was always the 4,5-6,7 group. Ten micromolar Cd2+ altered the sliding pattern similarly to Ni2+, with 28% two-sided extrusion, 55.9% one-sided extrusion and 16.1% with no extruded bundles.Either pretreatment regimen impeded extrusion of the 9,1,2 group in a high percentage of cells, compared to untreated cells. This specific inhibition of the 9,1,2 side by Ni2+ or Cd2+ is especially significant since Ni2+ also inhibits spontaneous wave initiation in bull sperm (Lindemann et al.: Journal of Cell Biology 87:420-426, 1980), and both Ni2+ and Cd2+ reportedly block the flagellar Ca2+-response in rat sperm (Lindemann and Goltz: Cell Motility and the Cytoskeleton 10:420-431, 1988; Lindemann et al.: Cell Motility and the Cytoskeleton 20:316-324, 1991). © 1993 Wiley-Liss, Inc.
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  • 19
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 20
    ISSN: 0886-9383
    Keywords: Calibration ; Non-linearity ; Principal components ; Stein estimate ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A new regression method for non-linear near-infrared spectroscopic data is proposed. The technique is based on a model which is linear in the principal components and simple functions (squares and products) of them. Added variable plots are used to determine which squares and products to incorporate into the model. The regression coefficients are estimated by a Stein estimate which shrinks towards the estimate determined by the first several principal components and the selected non-linear terms. The technique is not computationally intensive and is appropriate for routine predictions of chemical concentrations. The method is tested on three data sets and in all cases gives more accurate predictions than does linear principal components regression.
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  • 21
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 22
    ISSN: 0886-9383
    Keywords: Non-linear mapping ; Graphical methods ; SAR ; SPR ; Quality of representation ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: From a review of the theoretical aspects of non-linear mapping and the different algorithms available in the literature, the methodological and practical problems linked to the use of this multivariate method in structure-activity and structure-property relationship studies are underlined. Useful tools for the graphical display of the outputs and the interpretation of the obtained clusters are presented. Statistical parameters estimating the quality of the graphical representation of each individual are also introduced. An example of application on a data matrix of 37 acaricides described by four physicochemical descriptors (π, F, R, MR) is presented.
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  • 23
    ISSN: 0886-9383
    Keywords: Multicomponent analysis ; Factor analysis ; Detection limit ; Local rank ; Zero-component region ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In this paper we redefine the term detection limit to embrace the inherent multivariate nature of samples, instrumental measurements and chemometrics resolution procedures. The so-called zero-component regions, i.e. parts with no chemical components eluting, are used as repeated analytical blanks to estimate a statistical multivariate detection limit for determining the number of chemical species in local regions of a single two-way chromatogram or a collection of synchronized one-way chromatograms. For two-way chromatography the detection limit is determined from the distribution of the first eigenvalues obtained from all possible combinations of spectra in the zero-component regions. The number of spectra in each calculation should correspond to the number included in the later examination of the local retention time regions. For one-way chromatography on a collection of samples with similar chemical components at varying concentrations the same procedure is used, with the samples taking the role of the spectra in two-way chromatography. The detection limit can be chosen at various confidence levels depending on whether false positive or negative detection of minor components is most critical. The results obtained from the zero-eigenvalue distribution are more robust than those obtained by a previously developed F-test.
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  • 24
    ISSN: 0886-9383
    Keywords: Reaction kinetics ; Initial rate ; Kinetic order ; Response surface modelling ; Canonical analysis ; Organic synthesis ; Optimization ; Reaction mechanisms ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A method is presented by which it is possible to estimate the initial rate of chemical reactions when the experimental conditions are varied according to a response surface design. The method is intended as a complementary method for analysing data obtained from experiments in synthetic chemistry when the objective is to optimize the yield of the reaction.Data obtained by simulations have been used to develop the method. From the simulated reactions it is shown that sequential analysis of the chemical yield of the reaction makes it possible to estimate models which describe how the parameters of the response surface of the yield vary over time. The derivatives of these time functions of the response surface parameters can be used to define a rate function which describes how the variations in the experimental conditions influence the rate of the reaction.It is shown how such rate functions can be used to afford reasonable estimates of the initial rates of the reaction. The initial reaction rates thus estimated can be used to determine the kinetic order of the reactants and also to provide estimates of the activation energy of the reaction.A thorough discussion of how canonical analysis of the rate function may assist in the elucidation of reaction mechanisms is given.
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  • 25
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    Journal of Chemometrics 7 (1993), S. 439-445 
    ISSN: 0886-9383
    Keywords: Calibration ; Rank annihilation ; Residual bilinearization ; Three-way ; Trilinear ; Net analyte rank ; Second-order ; Generalized rank annihilation method (GRAM) ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Through theoretical analysis and computer simulation, this short communication comments on the residual bilinearization (RBL) method and compares it with non-bilinear rank annihilation (NBRA) for the treatment of second-order calibration with non-bilinear data. It is found that these two methods are mathematically equivalent but have different noise propagation properties. The second-order advantage, namely quantitation in the presence of unknown interferences, can be carried over to non-bilinear data only if there exists a net analyte rank (NAR) for the analyte of interest.
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  • 26
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 7 (1993), S. 455-475 
    ISSN: 0886-9383
    Keywords: Biological activity ; Cross-validation ; Exchangeability ; Molecular descriptors ; Prediction ; Relationship ; Structure ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The two parts of this paper form a critique of a variety of statistical techniques of actual or potential use in quantitative structure-activity relationship (QSAR) studies and related fields. Part I explores the statistical thinking that is needed to underpin those techniques. Emphasis as placed on (a) the role of ‘exchangeability’ as an alternative to unrealistic statistical modelling and (b) the use of cross-validation to limit self-deception in the use of any particular technique. The problem of the almost unlimited range of molecular descriptors is seriously addressed. (Part II provides a concise critical review of methods-some well-established and some new.)
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  • 27
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    Journal of Chemometrics 7 (1993), S. 305-340 
    ISSN: 0886-9383
    Keywords: Stem & leaf display ; Histogram ; Boxplot ; Quantile plot ; Scatterplot ; Regression ; Smoothing ; 3D rotation ; Scatterplot matrix ; OMEGA strategy ; Dimension reduction ; Stability of structure ; Resampling ; Interpretation of structure ; Prediction models ; Variables selection ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Exploratory data analysis (EDA) is a toolbox of data manipulation methods for looking at data to see what they seem to say, i.e. one tries to let the data speak for themselves. In this way there is hope that the data will lead to indications about ‘models’ of relationships not expected a priori. In this respect EDA is a pre-step to confirmatory data analysis which delivers measures of how adequate a model is. In this tutorial the focus is on multivariate exploratory data analysis for quantitative data using linear methods for dimension reduction and prediction. Purely graphical multivariate tools such as 3D rotation and scatterplot matrices are discussed after having introduced the univariate and bivariate tools on which they are based. The main tasks of multivariate exploratory data analysis are identified as ‘search for structure’ by dimension reduction and ‘model selection’ by comparing predictive power. Resampling is used to support validity, and variables selection to improve interpretability.
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  • 28
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    Journal of Chemometrics 7 (1993), S. 393-425 
    ISSN: 0886-9383
    Keywords: Preprocessing ; Closure ; Normalization ; Ratioing ; Constant sum transformation ; Constant length transformation ; Maximum value transformation ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The geometric properties of three common object-preprocessing transformations (constant sum, or closure; constant length, or normalization; and maximum value, or ratioing) are investigated. An argument is made for using absolute values in the constant sum and maximum value transformations. In general, each transformation distorts the shape and dimensionality of patterns in the data: transformed data lie on (C-1)-dimensional surfaces in the original C-dimensional space. A data set that has been closed by one of these transformations can be reopened if a vector containing the constant sums, constant lengths or maximum values of the original objects was retained. Transformed data sets may be freely interconverted among these three transformations without the loss of information.
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  • 29
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    Journal of Chemometrics 7 (1993), S. 447-452 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 30
    ISSN: 0887-3585
    Keywords: peptide chains ; lipid membranes ; Monte Carlo simulation techniques ; hydropathy scale method ; tripeptides ; phospholipid membranes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A combination of dynamic Monte Carlo simulation techniques with a hydropathy scale method for the prediction of the location of transmembrane fragments in membrane proteins is described. The new hydropathy scale proposed here is based on experimental data for the interactions of tripeptides with phospholipid membranes (Jacobs, R.E., White, S.H. Biochemistry 26:6127-6134, 1987) and the self-solvation effect in protein systems (Roseman, M.A., J. Mol. Biol. 200:513-522, 1988). The simulations give good predictions both for the state of association and the orientation of the peptide relative to the membrane surface of a number of peptides including Magainin2, M2δ, and melittin. Furthermore, for Pf1 bacterio-phage coat protein, in accord with experiment, the simulations predict that the C-terminus forms a transmembrane helix and the N-terminus forms a helix which is adsorbed on the surface of the bilayer. Finally, the present series of simulations provide a number of insights into the mechanism of insertion of peptides into cell membranes. © 1993 Wiley-Liss, Inc.
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  • 31
    ISSN: 0887-3585
    Keywords: aequorin ; photoprotein ; crystallization ; bioluminescence ; X-ray diffraction ; metal-binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 Å resolution. The crystals are orthorhombic, space group P21212 1; the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) Å. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis. © 1993 Wiley-Liss, Inc.
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  • 32
    ISSN: 0887-3585
    Keywords: ribonuclease H ; Thermus thermophilus HB8 ; DNA/RNA hybrid ; crystallization ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Ribonuclease H from an extreme thermophile, Thermus thermophilus HB8, has been crystallized from solutions at low ionic strength. The crystals belong to the hexagonal space group P6 122 (or P6 522), with unit cell parameters a = b = 44.7 Å, c = 314.7 Å. They contain one 18,000 Mr molecule per asymmetric unit and diffract to 2.8 Å resolution. © 1993 Wiley-Liss, Inc.
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  • 33
    ISSN: 0887-3585
    Keywords: protein simulations ; molecular dynamics ; serine protease ; enzyme-substrate interactions ; hydrogen bonding analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The binding free energy difference for the Gly-169 → Ala-169 (G169A) mutation in subtilisin BPN′ complexed with a tripeptide substrate analogue is explored using the thermodynamic integration approach. The structure of the mutant enzyme-substrate complex obtained from free energy simulation is in good agreement with experimental X-ray refinement. The near perfect reversibility is obtained in the present work for ensuring the correctness of the free energy calculations. The results of the binding free energy difference are close to similar experimental data. © 1993 Wiley-Liss, Inc.
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  • 34
    ISSN: 0887-3585
    Keywords: dogfish C-reactive protein ; vapor phase equilibration ; ammonium sulfate ; sitting drop technique ; hexamers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals of dogfish (Mustelus canis) C-reactive protein were obtained through vapor phase equilibration using the sitting drop rod technique with ammonium sulfate as the precipitating agent. The space group was determined to be P1 (triclinic lattice) with unit cell dimensions of a = 82.91, b = 92.25 and c = 103.40 Å; α = 83.36°, β = 89.76°, and γ = 81.30°. These crystals diffract to about 2.6 Å resolution and contain two hexamers in the asymmetric unit. © 1993 Wiley-Liss, Inc.
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  • 35
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    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 36
    ISSN: 0887-3585
    Keywords: electrostatics ; conformational energies ; solvation ; ionization ; force fields ; energy functions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Computer models of proteins frequently treat the energies and forces associated with ionizable groups as if they were purely electrostatic. This paper examines the validity of the purely electrostatic approach, and concludes that significant errors in energies can result from the neglect of ionization changes. However, a complete treatment of ionizable groups presents substantial computational obstacles, because of the large number of ionization states which must be examined in systems having multiple interacting titratable groups. In order to address this problem, two novel methods for treating the energetics and forces associated with ionizable groups with a minimum of computer time have been developed. The most rapid method yields approximate energies by computing the free energy of a single highly occupied ionization state. The second method separates ionizable groups into clusters, and treats intracluster interactions exactly, but intercluster interactions approximately. This method yields both accurate energies and fractional charges. Good results are obtained in tests of both methods on proteins having has many as 123 ionizable groups. The more rapid method requires computer times of 0.01 to 0.34 sec, while the more accurate method requires 0.7 to 15 sec. These methods may be fast enough to permit the incorporation of ionization effects in iterative computations, such as energy minimizations and conformational searches. © 1993 Wiley-Liss, Inc.
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  • 37
    ISSN: 0887-3585
    Keywords: lectin ; stinging nettle ; Urtica dioica ; crystallization ; X-ray diffraction ; protein-carbohydrate interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Single crystals were grown from affinity-purified stinging nettle lectin and from its complex with the specific trisaccharide NN′N″ -triacetylchitotriose by vapor diffusion at room temperature. The lectin crystallizes in space group P212121 with unit cell dimensions a = 54.3 (1) Å, b = 62.2 (1) Å, and c = 92.4 (2) Å, and diffracts to 3.0 Å resolution. The asymmetric unit contains three lectin monomers. The crystals of the lectin-trisaccharide complex have space group P212121 with cell constants a = 37.69 (4) Å, b = 48.97 (6) Å, and c = 57.32 (4) Å. These crystals diffract to at least 2.0 Å resolution and the asymmetric unit contains one lectin monomer. A three-dimensional X-ray structure determination is on its way. © 1993 Wiley-Liss, Inc.
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  • 38
    ISSN: 0887-3585
    Keywords: ROP ; 4-α-helix bundles ; protein stability ; protein crystallization ; calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.
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  • 39
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    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: calcium binding ; crystal structure ; protein stability ; site-directed mutagenesis ; subtilisin ; X-ray crystallography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A version of subtilisin BPN′ lacking the high affinity calcium site (site A) has been produced through genetic engineering methods, and its crystal structure refined at 1.8 Å resolution. This protein and the corresponding version containing the calcium A site are describedand compared. The deletion of residues 75-83 was made in the context of four site-specific replacements previously shown to stabilize subtilisin. The helix that in wild type is interrupted by the calcium binding loop, is continuous in the deletion mutant, with normal geometry. A few residues adjacent to the loop, principally those that were involved in calcium coordination, are repositioned and/or destabilized by the deletion. Because refolding is greatly facilitated by the absence of the Caloop, this proteinoffers a new vehicle for analysis and dissection of the folding reaction. This is among the largest internal changes to a protein to be described at atomic resolution. © Wiley-Liss, Inc.
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  • 40
    ISSN: 0887-3585
    Keywords: HIV-1 protease ; HIV-2 protease specificity ; 8-D space ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A vector projection method is proposed to predict the cleavability of oligopeptides by extended-specificity site proteases. For an enzyme with eight specificity subsites the substrate octapeptide can be uniquely expressed as a vector in an 8-dimensional space, whose eight bases correspond to the amino acids at the eight subsites, P1, P1′, P2′, P3′ and P4′, respectively. The component of such a characteristic vector on each of the eight bases is defined as the frequency of an amino acid occurring at a given site. These frequencies were derived from a set of octapeptides known to be cleaved by HIV protease. The cleavability of an octapeptide can then be estimated from the projection of its characteristic vector on an idealized, optimally cleavable vector. The high ratio of correct prediction vs. total prediction for the data in both the training and the testing sets indicates that the new method is self-consistent and efficient. It provides a rapid and accurate algorithm for analyzing the specificity of any multisubsite enzyme for which there is no coupling between subsites. In particular, it is useful for predicting the cleavability of an oligopeptide by either HIV-1 or HIV-2 protease, and hence offers a supplementary means for finding effective inhibitors of HIV protease as potential drugs against AIDS. © Wiley-Liss, Inc.
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  • 41
    ISSN: 0887-3585
    Keywords: enolase ; fluoride ; X-ray diffraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Enolase in the presence of its physiological cofactor Mg2+ is inhibited by fluoride and phosphate ions in a strongly cooperative manner (Nowak, T, Maurer, P. Biochemistry 20:6901, 1981). The structure of the quaternary complex yeast enolase-Mg2+-F--Pi has been determined by X-ray diffraction and refined to an R = 16.9% for those data with F/σ(F) ≥ 3 to 2.6 Å resolution with a good geometry of the model. The movable loops of Pro-35-Ala-45, Val-153-Phe-lo9, and Asp-255-Asn-266 are in the closed conformation found previously in the precatalytic substrate-enzyme complex. Calculations of molecular electrostatic potential show that this conformation stabilizes binding of negatively charged ligands at the Mg2+ ion more strongly than the open conformation observed in the native enolase. This closed conformation is complementary to the transition state, which also has a negatively charged ion, hydroxide, at Mg2+. The synergism of inhibition by F- and Pi most probably is due to the requirement of Pi, for the closed conformation. It is possible that other Mg2+-dependent enzymes that have OH- ions bound to the metalion in the transition state also will be inhibited by fluoride ions. © Wiley-Liss, Inc.
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  • 42
    ISSN: 0887-3585
    Keywords: thermodynamics ; calorimetry ; protein-hormone interaction ; drug design ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The ability to predict the strength of the association of peptide hormones or other ligands with their protein receptors is of fundamental importance in the fields of protein engineering and rational drug design. To form a tight complex between a flexible peptide hormone and its receptor, the large loss of configurational entropy must be overcome. Recently, the crystallographic structure of the complex between angiotensin II and the Fab fragment of a high affinity monoclonal antibody has been determined (Garcia, K. C., Ronco, P. M., Verroust, P. J., Brünger, A. T., Amzel, L. M. Three-dimensional structure of an angiotensin II-Fab complex at 3 Å: Hormone recognition by an anti-idiotypic antibody. Science 257:502-507, 1992). In this paper we present a study of the thermodynamics of the association by high sensitivity isothermal titration calorimetry. The results of the experiments indicate that at 30°C the binding is characterized by (1) a ΔH of -8.9 ± 0.7 kcal mol-1, (2) a ΔCp of -240 ± 20 cal K-1 mol-1, and (3) the release of 1.1 ± 0.1 protons per binding site in the pH range 6.0-7.3. Using these values and the previously determined binding constant in phosphate buffer, ΔG at 30°C is estimated as -11 kcal mol-1 and ΔS as 6.9 cal K-1 mol-1. The calorimetric data indicate that binding is favored both enthalpically and entropically. These results have been complemented by structural thermodynamic calculations. The calculated and experimentally determined thermodynamic quantities are in good agreement. Entropically, the loss of configurational entropy is more than compensated by the entropy gain from solvent release associated with the hydrophobic effect. Enthalpically, binding is favored by polar interactions (hydrogen bonding). Consequently, the problem of binding flexible hormones is solved in much the same way as the folding of an unstructured polypeptide chain into a globular protein. © 1993 Wiley-Liss, Inc.
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  • 43
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    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: X-ray crystallography ; carbonic anhydrase ; cyanide ; cyanate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Carbonic anhydrase is inhibited by the “metal poison” cyanide. Several spectroscopic investigations of carbonic anhydrase where the natural zinc ion has been replaced by cobalt have further strengthened the view that cyanide and cyanate bind directly to the metal. We have determined the structure of human carbonic anhydrase II inhibited by cyanide and cyanate, respectively, by X-ray crystallography. It is shown that the inhibitors replace a molecule of water, which forms a hydrogen bond to the peptide nitrogen of Thr-199 in the native structure. The coordination of the zinc ion is hereby left unaltered compared to the native crystal structure, so that the zinc coordinates three histidines and one molecule of water or hydroxyl ion in a tetrahedral fashion. The binding site of the two inhibitors is identical to what earlier has been suggested to be the position of the substrate (CO2) when attacked by the zinc bound hydroxyl ion. The peptide chain undergoes no significant alterations upon binding of either inhibitor. © 1993 Wiley-Liss, Inc.
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  • 44
    ISSN: 0887-3585
    Keywords: crystallization ; antilysozyme system ; epitope ; antigen-antibody complex ; cross-reactivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The complex formed between the Fab fragment of a murine monoclonal anti-hen egg lysozyme antibody F9.13.7 and the het-erologous antigen Guinea-fowl egg lysozyme has been crystallized by the hanging drop technique. The crystals, which diffract X-rays to 3 Å resolution, belong to the monoclinic space group P21, with a = 83.7 Å, b = 195.5 Å, c = 50.2 Å, β = 108.5° and have two molecules of the complex in the asymmetric unit The three-dimensional structure has been determined from a preliminary data set to 4 Å using molecular replacement techniques. The lysozyme-Fab complexes are arranged with their long molecular axes approximately parallel to the crystallo-graphic unique axis. Fab F9.13.7 binds an anti-genie determinant that partially overlaps the epitope recognized by antilysozyme antibody HyHEL10. © 1993 Wiley-Liss, Inc.
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  • 45
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    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: leucine zipper ; protein folding ; molecular dynamics ; skip residue ; hemaglutinin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Two complementary methods for measuring local pitch based on heptad position in α-helical coiled coils are described and applied to six crystal structures. The results reveal a diversity of pitch values: two-stranded coiled coils appear to have pitch values near 150 Å the values for three- and four-stranded coiled coils range closer to 200 Å. The methods also provide a rapid and sensitive gauge of local coiled-coil conformation. Polar or charged residues in the apolar interface between coiled-coil helices markedly affect local pitch values, suggesting a connection between pitch uniformity and coiled-coil stability. Moreover, the identification of a skip residue (heptad frame shift) in the hemaglutinin glycoprotein of influenza virus (HA) allows interpretation of local pitch changes. These results on relatively short coiled-coil structures have relevance for the much longer fibrous proteins (many of which have skip residues) whose detailed structures are not yet established. We also show that local pitch values from molecular dynamics predictions of the GCN4 leucine zipper are in striking agreement with the high-resolution crystal structure - a result not readily discerned by direct comparison of atomic coordinates. Taken together, these methods reveal specific aspects of coiled-coil structure which may escape detection by global analyses of pitch. © 1993 Wiley-Liss, Inc.
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  • 46
    ISSN: 0887-3585
    Keywords: phosphoglycerate kinase ; Bacillus stearothermophilus ; crystallographic structure ; thermal stability ; increased hydrophobicity ; helix stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast. The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures. The refinement residual, calculated using all the measured data between 8 and 1.65 Å, is 0.18(1). The stereo chemical deviations of the final model from ideality are 0.01 Å for both bonds and planes.The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53°C, respectively. Differential scanning calorimetry indicates that the energy difference (ΔΔG) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol-1 at room temperature. The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better α-helix stability resulting from the removal of helix destablising residues and extra helix-dipole/helix side chain ionic interactions. © 1993 Wiley-Liss, Inc.
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  • 47
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 48
    ISSN: 0887-3585
    Keywords: AIDS ; energy minimization ; enzyme inhibition ; molecular modeling ; protein conformation ; crosscorrelation map ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The dynamic behavior of one 99-residue subunit of the dimeric aspartyl protease of HIV-1 was studied in a 160 psec molecular dynamics simulation at 300 K in water. The crystal structure of one of the identical subunits of the dimer was the starting point, with the aqueous phase modeled by 4,331 explicit waters in a restrained spherical droplet Analysis of the simulations showed that the monomer displayed considerable flexibility in the interfacial portions of the flap (the region which folds over the substrate), the N- and C-0termini, and, to a lesser extent, the active site. The flap undergoes significant motion as an independent rigid finger, but without the cantilever previously reported hi a simulation of the dimer. The N-terminus displayed the greatest fluctuational disorder whereas the C-terminus exhibited the greatest root mean square movement from the crystal structure. The central core of the monomer had a heavy-atom root mean square deviation from the initial structure of about 3.0 Å during the latter half of the simulation. Although this is larger than the 1.6 Å found for comparable simulations of typical globular proteins, the general features of the tertiary structure were preserved over the course of the simulation. Overall, these results indicate that the relaxed structure obtained in these simulations may provide a better model for the tertiary structure of the solvated HIV-1 protease monomer than the subunit conformation seen in the X-ray crystallographic structure of the dimer. Except in the flap region, the design of compounds intended to interfere with dimerization should take this relaxation and the flexibility of the solvated monomer, especially at the termini, into account. © 1993 Wiley-Liss, Inc.
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  • 49
    ISSN: 0887-3585
    Keywords: computer simulation ; side chain conformations ; optimization ; α-helices ; tertiary structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present a novel search strategy for determining the optimal packing of protein secondary structure elements. The approach is based on conformational energy optimization using a predetermined set of side chain rotamers and appropriate methods for sampling the conformational space of peptide fragments having fixed backbone geometries. An application to the 4-helix bundle of myohemerythrin is presented. It is shown that the conformations of the amino acid side chains are largely determined at the level of helix pairs and that superposition of these results can be used to construct the full bundle. The final solution obtained, taking into account restrictions due to the lateral amphiphilicity of the helices, differs from the native structure by only a 20° rotation of a single helix. © 1993 Wiley-Liss, Inc.
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  • 50
    ISSN: 0887-3585
    Keywords: tryptophan ; fluorescence ; phosphorescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: In order to correlate between spectroscopic and structural changes in a protein, the environment of Trp 135 in T4 lysozyme was deliberately perturbed by the replacement of Gln 105 with alanine (Q105A), glycine (Q105G), and glutamic acid (Q105E). In wild-type lysozyme, Trp 135 is buried, but the indole nitrogen is hydrogen-bonded to the side-chain of Gln 105. In the Q105G and Q105A mutant structures, the indole nitrogen becomes accessible to solvent. Crystallographic analysis shows that the structures of all of the mutants are similar to wild-type. There are, however, distinct rearrangements of the local solvent structure in response to the new side-chains. There are also small but significant changes in the relative orientations of the two domains of the protein that appear to result from a series of small, concerted movements of side-chains adjacent to residue 105. Evaluation of the fluorescence and phosphorescence of the mutant proteins in terms of their observed three-dimensional structures shows that large spectral changes do not necessarily imply large changes in structure or in static solvent accessibility. Increases in polar relaxation about the excited state of tryptophan may be the result of only small increases in local dynamics or solvent exposure. 1H-NMR was also used to monitor the effects of the substitutions on Trp 138. In Q105E, but not in Q105G, Q105A and WT, the Hε1 chemical shift of Trp 138 is very pH-dependent, apparently reflecting the titration of Glu 105 which has a spectroscopically determined pKa of 6.0. The elevation of the pKa of Glu 105 in Q105E is also reflected in the pH dependence of the stability of this mutant. © 1993 Wiley-Liss, Inc.
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    ISSN: 0887-3585
    Keywords: molecular dynamics ; free energy ; perturbation theory ; kinetic mechanism ; dissociation constants ; dihydrofolate reductase ; 8-methyl-pterins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecular dynamics simulation and free energy perturbation techniques have been used to study the relative binding free energies of the designed mechanism-based pterins, 8-methylpterin and 6,8-dimethylpterin, to dihydrofolate reductase (DHFR), with co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The calculated free energy differences suggest that DHFR.NADPH.6,8-dimethylpterin is thermodynamically more stable than DHFR.NADPH.8-methylpterin by 2.4 kcal/mol when the substrates are protonated and by 1.3 kcal/mol when neutral. The greater binding strength of 6,8-dimethylpterin may be attributed largely to hydration effects. In terms of an appropriate model for the pH-dependent kinetic mechanism, these differences can be interpreted consistently with experimental data obtained from previous kinetic studies, i.e., 6,8-dimethylpterin is a more efficient substrate of vertebrate DHFRs than 8-methylpterin. The kinetic data suggest a value of 6.6 ± 0.2 for the pKa of the active site Glu-30 in DHFR.NADPH. We have also used experimental data to estimate absolute values for thermodynamic dissociation constants of the active (i.e., protonated) forms of the substrates: these are of the same order as for the binding of folate (0.1-10 μM). The relative binding free energy calculated from the empirically derived dissociation constants for the protonated forms of 8-methylpterin and 6,8-dimethylpterin is 1.4 kcal/mol, a value which compares reasonably well with the theoretical value of 2.4 kcal/mol. © 1993 Wiley-Liss, Inc.
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  • 52
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: antigen-antibody recognition ; docking algorithm ; distance-dependent dielectric ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A Monte Carlo algorithm that searches for the optimal docking configuration of hen egg white lysozyme to an antibody is developed. Both the lysozyme and the antibody are kept rigid. Unlike the work of other authors, our algorithm does not attempt to explicitly maximize surface contact, but minimizes the energy computed using coarse-grained pair potentials. The final refinement of our best solutions using all-atom OPLS potentials (Jorgensen and Tirado-Rives8) consistently yields the native conformation as the preferred solution for three different antibodies. We find that the use of an exponential distance-dependent dielectric function is an improvement over the more commonly used linear form. © 1993 Wiley-Liss, Inc.
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  • 53
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: heavy chains ; complementarity determining region ; antibody specificity ; amino acid loops ; V-(D)-J joining ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Sequences of the third complementarity determining region of antibody heavy chains (CDRH3s) are listed according to their length. Human sequences vary from 2 to 26 amino acids residues, but less extensively in other species. When combined with the other five complementarity determining regions, this enormous length variation of CDRH3, together with amino acid substitutions in their sequences, can provide a very large number of antibody specificities and can influence the shape of antibody combining sites.©1993 Wiley-Liss,Inc.
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  • 54
    ISSN: 0887-3585
    Keywords: pattern recognition ; structure-activity data base ; receptor binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A novel computer modeling approach suitable for the structure analysis of small bioactive peptides has been developed. This approach involves identification of conformational patterns in protein structure data bank based on the sequence homology with the bioactive peptide. The models built on the basis of this homology and having common conformational patterns are analyzed under the structural constraints derived from the activity data of various synthetic analogs of the peptide. Application of this procedure to the gonadotropin releasing hormone (GnRH) resulted in a library of possible structures for GnRH, 9 among which shared a common β-turn. Further analysis of the structures containing the β-turn motif, in the context of the structure-activity data, led to a model for the active conformation of GnRH. The topology of the putative receptor binding site of the hormone is defined by a contiguous surface formed through an appropriate juxtaposition of the N-terminal pGlu1 the guanidyl group of Arg8, aromatic side chain of Trp3, and the Gly10-NH2 at the C-terminal end. ©Wiley-Liss, Inc.
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  • 55
    ISSN: 0887-3585
    Keywords: X-ray crystallography ; antibody domain ; recombinant DNA ; binding affinity ; antigen-antibody complex ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P4212, with unit cell dimensions a = b = 141 Å, c = 218 Å. © 1993 Wiley-Liss, Inc.
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  • 56
    ISSN: 0887-3585
    Keywords: trp-repressor ; TIP3P water ; hydrophobic shell ; hydrogen bond ; AMBER ; diffusion coefficient ; radial distribution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The solvent structure and behavior around a protein were examined by analyzing a trajectory of molecular dynamics simulation of thetrp-holorepressor in a periodic box of water. The calculated selfdiffusion coefficient indicated that the solvent within 10 Å of the protein had lower mobility. Examination of the solvent diffusion around different atoms of different kinds of residues showed no general tendency. Thisfact suggested that the solvent mobility is not influenced significantly bythe kind of the atom or residue they solvated. Distribution analysis aroundthe protein revealed two peaks of water oxygen: a sharp one at 2.8 Å around polar and charged atoms and a broad one at ∼3.4 Å aroundapolar atoms. The former was stabilized by water-protein hydrogen bonds, and the latter was stabilized by water-lwater hydrogen bonds, suggesting the existence of a hydrophobic shell. An analysis of protein atom-water radial distribution functions confirmed these shell structures around polar or charged atoms and apolar ones. © 1993 Wiley-Liss, Inc.
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