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  • Wiley-Blackwell  (12,136)
  • 1990-1994  (12,136)
  • 1970-1974
  • 1965-1969
  • 1991  (12,136)
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  • 1990-1994  (12,136)
  • 1970-1974
  • 1965-1969
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  • 1
    ISSN: 0886-1544
    Keywords: β-tubulin ; CHO ; taxol ; colcemid ; drug resistance ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: LY195448 is an experimental drug that blocks cells at metaphase (Boder et al.:Microtubules and Microtubule Inhibitors 1985: 353-361, 1985). A 4 hour exposure of NRK cells to a drug concentration of 46 μM (15 μg/ml) increased the number of mitotic cells in the population from 4.9% to 18.5%. Examination of treated cells by immunofluorescence showed increased numbers of cells blocked at prometaphase, with short microtubules extending from the spindle pole to the kinetochores. The cytoskeleton of interphase cells remained intact at these concentrations. However, the number of microtubules appeared to be reduced, and those that remained appeared kinkier and curled, particularly toward the periphery of the cells. When cytoskeletal microtubules of NRK cells were depolymerized with nocodazole, they reassembled within minutes of transfer to drug-free media. However, nocodazole-treated cells transferred to fresh media containing 15 μg/ml of LY 195448 required 2-3 times longer to reassemble cytoplasmic microtubules. Previously isolated Chinese hamster ovary cell microtubule mutants resistant to either taxol or Colcemid were tested for cross-resistance to this drug. Cell lines resistant to the depolymerizing drug Colcemid exhibited increased resistance to LY 195448 compared to wild-type cells, whereas taxol resistant cell lines were more sensitive. Of eleven newly isolated mutant CHO cell lines selected for increased resistance to LY 195448, seven exhibited an altered β-tubulin protein by two-dimensional polyacrylamide gel electrophoresis. These 11 cell lines also showed a heterogenous pattern of resistance to several microtubule-active drugs. These data demonstrate that LY 195448 is cytotoxic to mammalian cells because it inhibits microtubule assembly, most likely through a direct interaction with tubulin.
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  • 2
    ISSN: 0886-1544
    Keywords: Fusarium ; mitosis ; mitotic mechanisms ; motion analysis ; video microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Forces that elongate the spindle during anaphase B of mitosis might be generated in the asteis, in the spindle, or in both. In the fungus Nectria haematococca, it has already been shown that the asters pull on the spindle pole bodies (SPBs) through-out anaphase B. In this study, we used computerized video motion analysis to characterize brief episodes of spindle bending and straightening to find out if such bending is caused by spindle pushing forces. In three episodes there were two distinct components of spindle bending and straightening: one spanning the entire episode and comprising spindle elongation and another, superimposed on the first, involving a shortening of the distance between the SPBs. In a fourth episode, only spindle elongation was involved. All four spindles elongated rapidly while bending and underwent net growth during the overall bending-straightening episode at an average rate of 4.2 μm/min. The path of one aster of a fifth mitotic apparatus was blocked by a large, occluding vacuole. This obstacle caused the migration of the mitotic apparatus to stop, resulting in a long (25 sec) episode of spindle curving and bending, usually without any substantial reduction in the distance between the SPBs as well as a marked reduction (from 4.7 to 0.65 μm/min) in the rate of spindle elongation. The results provide evidence that spindle pushing forces are active in vivo during anaphase B in N. haematococca and that they, along with astral pulling forces, help to elongate the spindle at a mostly constant rate. This is the first demonstration of both kinds of spindle elongation forces in the same organism.
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  • 3
    ISSN: 0886-1544
    Keywords: immunofluorescence ; Rh-ph ; mitosis ; cytochalasin B ; stomates ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Actin localization during stomatal complex formation in rye leaf epidermis was compared by three different labeling procedures. When leaf segments are fixed with formaldehyde prior to staining microfilament (MF) patterns visualized with actin antibodies and those with rhodamine-phalloidin (Rh-ph) are basically identical in controls. Likewise, on tissues treated with cytochalasin B (CB), actin antibodies and Rh-ph produce very similar labeling patterns. Compared to MF alignments in fixed samples, additional sets of MFs are observed at the very cortical regions of epidermal cells that are stained with Rh-ph without aldehyde fixation. Cortical MFs are also present in a variety of mitotic cells; MFs of meristematic cells and guard mother cells are more concentrated near the walls facing spindle poles, whereas a fine meshwork of MFs is observed along the entire periclinal surface of subsidiary mother cells. Although exactly how MFs are involved in control of the division site in higher plant cells is still to be determined, the presence of MFs during mitosis and the abnormal division observed in some stomatal cells after treatment with CB suggest that MFs are necessary for normal orientation of division in these cells, and thus normal morphogenesis.
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  • 4
    ISSN: 0886-1544
    Keywords: microtubules ; invertebrate MAPs ; immunohistochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have identified a 260 kD polypeptide as being the major microtubule-associated protein (MAP) in the ovaries of the hemipteran insect, Oncopeltus fasciatus. The 260 kD insect ovarian MAP resembles certain mammalian brain MAPs by remaining soluble after boiling and promoting the assembly of tubulin into microtubules. It differs from most MAPs by exhibiting nucleotide-sensitivity, being removed from microtubules by both ATP and GTP. Antibodies specific for the 260 kD MAP allowed its immmunofluorescent localization to the massive micro-tubule aggregates forming the translocation systems which in hemipterans link the developing oocytes with anteriorly positioned nutritive cells. Such antibodies, in conjunction with electrophoretic methods, also demonstrated the 260 kD MAP to be species- and, to an extent at least, tissue-specific. The Oncopeltus 260 kD MAP was not present in the ovaries of either Notonecta or Corixa, hemipterans which have similar microtubule systems to Oncopeltus but MAPs of slightly different molecular weight. The 260 kD MAP from the ovaries of Oncopeltus was not present in neuronal ganglia of the same species. The significance of the species- and tissue-specificity of the 260 kD MAP, as well as its nucleotide-sensitivity, are speculated upon and discussed.
    Additional Material: 10 Ill.
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  • 5
    ISSN: 0886-1544
    Keywords: actin ; membrane cytoskeleton ; acrosome reaction ; DNase-1 ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Thyone sperm undergo an explosive acrosome reaction resulting in the extension of a 90 μm long acrosomal process. In unreacted sperm, profilamentous actin is sequestered within the profilactin cup (Tilney: Journal of Cell Biology 69:73-89, 1976), which consists of four major polypeptides: actin, profilin, and a 250/235 kDa equimolar doublet (TS 250/235). Dialysis of profilactin preparations into an actin assembly buffer resulted in the formation of acrosomal-like macromolecular aggregates containing actin, TS 250/235, and several other polypeptides as detected by SDS-PAGE. TS 250/235 was purified by subjecting extracts of pH solubilized profilactin cups to DEAE and phosphocellulose ion exchange chromatography. TS 250/235 demonstrated immunocrossreactivity with affinity purified polyclonal antibodies raised against S. purpuratus egg spectrin. As determined by biotinylated-calmodulin overlays, both subunits of TS 250/235 bound calmodulin in a Ca++-sensitive manner. Electron microscopy of low angle, rotary shadowed replicas of TS 250/235 revealed an elongate rod-shaped molecule with an average contour length of 203 nm. By indirect immunofluorescence, TS 250/235 was found to be uniformly distributed throughout the profilactin cup of the unreacted sperm. This distribution of TS 250/235 correlated with the location of monomeric actin as determined by localization studies utilizing fluorescent-DNase-1. Upon sperm activation, the cellular distribution of TS 250/235 dramatically changed and was observed both along the length and at the base of the extended acrosomal process.
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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
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 19 (1991), S. 290-290 
    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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  • 8
    ISSN: 0886-1544
    Keywords: video-enhanced contrast microscopy ; colcemid ; lamellipodia ; mitochondria ; intermediate filaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: It is known that depolymerization of microtubules by colcemid or other similar drugs abolishes polarization of pseudopodial activity in migrating fibroblasts. In this work the effect of colcemid on the intensity of protrusion and retraction of lamellipodia at the active edges of human fibroblasts migrating into the wound was investigated with video-enhanced contrast microscopy. To characterize the pseudopodial activity quantitatively the outlines of the active edges in the pairs of frames taken at adjacent 20-sec intervals were compared and mean areas of protrusions and retractions per unit length of the perimeter of the edge were measured. The mean rates of protrusions and retractions were 4-6 times less in colcemid-treated cells than in controls. Thus, microtubules depolymerized by colcemid, and/or intermediate filaments undergoing perinuclear collapse in the presence of this drug, are essential not only for the restriction of pseudopodial activity to one particular zone of the cell edge but also for the development of maximal activity in this zone.
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  • 9
    ISSN: 0886-1544
    Keywords: cytoskeleton ; morphology ; polymorphonuclear leukocytes ; human neutrophils ; scanning electron microscopy ; cytochalasins ; formyl peptides ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Neutrophils change shape from round to polar and sequentially polymerize/depolymerize actin following chemotactic peptide activation in suspension. To study the relationship between changes in F-actin content and shape we altered the kinetics/extent of actin polymerization and depolymerization with tBOC peptide, cytochalasin D (CD), and low-dose FMLP, and determined the effect of these alterations on the temporal sequence of changes in neutrophil shape. F-actin was measured by FACS analysis of NBDphallacidin-stained cells and expressed as relative fluorescent intensity (RFI) compared to control (RFI = 1.00). Shape was determined by scanning electron microscopy. FMLP causes serial polymerization/depolymerization of actin (RFI = 1.00 ± 0.04, 1.60 ± 0.21, 1.10 ± 0.18, and 1.05 ± 0.14) associated with four distinct shapes (round-smooth, round-ruffled, blebbed, and polar) noted at 0, 30, 90, 300 sec respectively. Since blebbed and polar shapes appear concurrent with depolymerization and following polymerization, we determined whether depolymerization is required for polarization of cells. The kinetics of depolymerization were: (1) accelerated by tBOC addition at 45 sec, and (2) slowed by high concentrations of FMLP (〉10-7 M) (300 sec RFI = 1.46). Neither change altered the time course of shape change. To determine whether duration of actin polymerization defines shape, polymerization was halted by addition of tBOC at 5, 10, 20, 30 sec after FMLP to block actin polymerization and shape was monitored at 300 sec. TBOC added 5-20 sec after FMLP limited neutrophil shape change to the blebbed form, while tBOC addition 30 sec following FMLP resulted in a polar shape at 300 sec. To determine whether the extent of actin polymerization affects the shape change sequence, polymerization was limited by (1) inhibition of polymerization with CD, (2) exposure of cells to low concentrations of FMLP ( 〈 10-9 M), and (3) interruption of polymerization with tBOC. Actin polymerization to RFI 〈 1.35-fold basal results in blebbed shape; polymerization 〉 1.35-fold basal yields polar shape. The data show: (1) the human neutrophil demonstrates intermediate shapes when activated by chemotactic peptide, (2) depolymerization of F-actin does not determine shape, and (3) blebbed shape appears when actin polymerizes for 〉5 sec; polar shape with polymerization ≥30 sec to RFI 〉 1.35-fold basal. The data suggest actin polymerization is required for, and extent of polymerization determines, the shape of human neutrophils.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 19 (1991), S. 221-222 
    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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  • 11
    ISSN: 0886-1544
    Keywords: spermatozoa ; cilia and flagella ; mechanochemical transduction ; dynein ; cell motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In the absence of glycolytic support, mammalian sperm derive their energy for motility from a densely packed array of mitochondria at the base of the flagellum known as the midpiece. Using data on the morphometric dimensions of over 200 mammalian species, we found that an allometric relationship exists between midpiece length (Lm) and flagellum length (Lf). Specifically, the length of the midpiece varies approximately as the 3/2 power of the flagellar length although the proportionality constant is different for eutherian and marsupial sperm. In contrast, when we corrected for the fraction of the midpiece that was taken up by mitochondria, a single linear correlation between mitochondrial volume and flagellar length for all mammals was found. These allometric relationships were used along with basic flagellar hydrodynamic theories to establish a unifying equation that predicted flagellar frequencies for any mammalian sperm between 40 μm and 200 μm in length. These findings imply that, at least in mammals, the mechanisms for energy production and dissipation in sperm flagella are highly conserved.
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  • 12
    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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  • 13
    ISSN: 0886-1544
    Keywords: centrin ; centrosome ; pericentriolar lattice ; pericentriolar material ; mitosis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In this study, we follow changes in localization of the centrin-related 165,000-Mr protein of PtK2 cells during the cell cycle. This protein is a component of a pericentriolar lattice that consists of pericentriolar satellites, pericentriolar matrix, and basal feet (Baron A.T., and J.L. Salisbury, J. Cell Biol. 107:2669-2678, 1988). By immunofluorescence microscopy, the 165,000-Mr protein is seen as a constellation of pericentrosomal spots. We observe that cells in late G1 and S are characterized by a dense centrosomal focus of spots with additional spots dispersed throughout the cytoplasm. In G2, one bright centrosomal focus of clustered spots is observed. As the cells proceed through prophase this single focus divides, forming two foci that move toward opposite sides of the nucleus. During prometaphase, each polar focus of spots disperses. At metaphase, the spots are distributed throughout each half-cytoplast from the poles to the chromosomes. During anaphase chromosome movement, some spots are seen beside and behind the trailing chromosome arms while others are clustered at the poles. At telo-phase, pericentrosomal spots radiate from the poles to surround each mass of chromatin. In early G1, pericentrosomal spots surround each newly formed nucleus. We conclude that the 165,000-Mr protein is a dynamic component of both the centrosome (pericentriolar matrix) and the mitotic apparatus (spindle matrix).
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  • 14
    ISSN: 0886-1544
    Keywords: cytoskeleton ; microfilaments ; immunocytochemistry ; photoreceptors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Actin has many diverse functions in the outer retina. To help elucidate its organization in this area, we have investigated the extent of its association with the actin cross-linking protein alpha-actinin. Ultrathin sections of chicken retina were double-immunolabelled with monospecific antibodies against actin and alpha-actinin. The highest relative amount of alpha-actinin to actin label was measured in the adherens junctions between the individual retinal pigmented epithelial (RPE) cells and between the photoreceptor and Mueller cells; in the photoreceptor myoid; and in the RPE basal microvilli. The lowest amount was in the Mueller cell microvilli, the RPE apical processes, and in the photoreceptor ellipsoid. It is likely that the areas containing the highest ratio of alpha-actinin to actin labelling are where the actin filaments are most highly cross-linked into bundles and linked to the plasma membrane by alpha-actinin. Actin filaments terminate in these areas, and, except for the myoid region, they are involved in cell-cell or cell-substrate adherens junctions.
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  • 15
    ISSN: 0886-1544
    Keywords: vimentin ; phosphatase inhibitors ; intermediate filaments ; stress fibers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Calyculin-A, an inhibitor of type 1 and 2A phosphatases, was applied extracel-lularly to 3T3 fibroblasts. At 0.1 μM, calyculin-A caused a marked increase in protein phosphorylation in both the cytosolic and insoluble cellular fractions. This effect was independent of external Ca2+. An immunoprecipitate, formed with an antibody to myosin, contained several cytoskeletal components. Increased phos-phorylation following treatment with calyculin-A was observed in vimentin, the 20-kD myosin light chain, and an unidentified 440-kD component. An enhanced level of vimentin phosphorylation was found in intermediate filament preparations from treated cells.Calyculin-A also caused marked shape changes of 3T3 cells. Within minutes after addition of calyculin-A (0.1 μM) cells became rounded and lost attachment to the substratum. Stress fibers, intermediate filaments, and microtubules, prominent in the attached control cells, were not evident in the rounded cells. Shape changes were reversible and after removal of calyculin-A the rounded cells attached to the substratum, resumed a flattened shape, and were active mitotically. In the cells treated with calyculin-A an unusual “ball-like” structure was observed with transmission electron microscopy. This unique structure was 2-3 μM in diameter and was located close to the nucleus.The use of calyculin-A adds further support to the idea that cell shape is controlled, at least in part, by concerted actions of a kinase-phosphatase couple.
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  • 16
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 17
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 5 (1991), S. 69-69 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 18
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 5 (1991), S. 121-128 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 19
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 5 (1991), S. 129-145 
    ISSN: 0886-9383
    Keywords: Multivariate calibration ; Biased regression ; Partial least squares (PLS) ; Principal component regression (PCR) ; Model validation ; Non-linear calibration ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: With the goal of understanding global chemical processes, environmental chemists have some of the most complex sample analysis problems. Multivariate calibration is a tool that can be applied successfully in many situations where traditional univariate analyses cannot. The purpose of this paper is to review multivariate calibration, with an emphasis being placed on the developments in recent years. The inverse and classical models are discussed briefly, with the main emphasis on the biased calibration methods. Principal component regression (PCR) and partial least squares (PLS) are discussed, along with methods for quantitative and qualitative validation of the calibration models. Non-linear PCR, non-linear PLS and locally weighted regression are presented as calibration methods for non-linear data. Finally, calibration techniques using a matrix of data per sample (second-order calibration) are discussed briefly.
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  • 20
    ISSN: 0886-9383
    Keywords: Digital filtering ; Real-time analysis ; Kalman filtering ; Infrared spectroscopy ; Principal components regression ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Real-time monitoring of pollutant levels from a mobile measuring platform requires fast, flexible data analysis methods. This paper reports a method for rapid analysis of passive remotely sensed infrared data with the aid of a Kalman filter. The background spectra produced by emission from the atmosphere are modelled at the start of the data collection sequence with a simple principal components model obtained by eigenanalysis of the initial ‘blank’ data taken with the spectrometer. The species of interest are included in the state space model by a separate measurement of their infrared spectra. It is demonstrated that for best filter performance in detecting the simulated pollutant species SF6 in the atmosphere, a filter model with two principal components describing the emission background works best. The filter ‘maps’ of SF6 closely follow the integrated spectral intensities measured after removal of suitable backgrounds.
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  • 21
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 5 (1991), S. 211-225 
    ISSN: 0886-9383
    Keywords: Shot noise ; Expectation-maximization ; Regression ; Deconvolution ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A simple algorithm for deconvolution and regression of shot-noise-limited data is illustrated in this paper. The algorithm is easily adapted to almost any model and converges to the global optimum. Multiple-component spectrum regression, spectrum deconvolution and smoothing examples are used to illustrate the algorithm. The algorithm and a method for determining uncertainties in the parameters based on the Fisher information matrix are given and illustrated with three examples. An experimental example of spectrograph grating order compensation of a diode array solar spectroradiometer is given to illustrate the use of this technique in environmental analysis. The major advantages of the EM algorithm are found to be its stability, simplicity, conservation of data magnitude and guaranteed convergence.
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  • 22
    ISSN: 0886-9383
    Keywords: Mean ; Variance ; Lognormal ; Optimal estimators ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Theoretical and simulation results are employed to evaluate mean and variance estimates for normal data when a lognormal distribution is assumed and for lognormal data when a normal distribution is assumed. Misspecifying the distribution leads to the use of suboptimal estimation methods. However, the results show that the suboptimal methods still produce estimators of good quality (low bias and variance) relative to the minimum variance unbiased estimators for each distribution, at least when practical efficiency is considered.
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  • 23
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 5 (1991), S. 249-261 
    ISSN: 0886-9383
    Keywords: Asymptotic power ; Clean-up standard ; Gamma distribution ; Likelihood ratio test ; Uniformly most powerful unbiased test ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The object of this paper is to develop a suitable statistical procedure to evaluate clean-up standards at hazardous waste sites. Under the assumptions that contaminant masses at a site follow a gamma distribution and that the data from the pre-remediation baseline sample as well as from the interim or final sample taken after a certain period of operation are both distributed as gamma with the same shape parameter but different scale parameters, we derive a uniformly most powerful unbiased test of the hypothesis that a specified percentage of contaminant mass has been reduced. A large-sample approximation of the exact test procedure and a comparison with the likelihood ratio test are provided.
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  • 24
    ISSN: 0886-9383
    Keywords: Correspondence analysis ; Eastern Lake Survey - Phase I data ; Acidic deposition ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Correspondence analysis (CA) was applied to lakewater data in order to study the effects of acidic deposition on the geochemical composition of lakes in the Adirondacks. The lake chemistry data analyzed were taken from the Eastern Lake Survey - Phase I (ELS-I) conducted by the U.S. Environmental Protection Agency. CA was used to identify ‘outlying’ lake samples as well as ‘superflous’ and ‘unresolved’ analytes. Correlational relationship among analytes were also examined.
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  • 25
    ISSN: 0886-9383
    Keywords: Errors in variables ; Orthogonal regression ; Latent variables ; Acid rain ; Acidic deposition ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Techniques for testing for and estimating relative bias between two laboratories are developed and applied to a survey of the chemistry of streams in the United States. The design of the quality assurance program allows estimation of linear corrections for bias as well as testing of the hypothesis of linearity. Designs of this type are useful, but improvements are suggested.
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  • 26
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 5 (1991), S. i 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 27
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 5 (1991), S. 487-501 
    ISSN: 0886-9383
    Keywords: Calibration ; Locally linear models ; Discrimination ; Optimality ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A calibration situation is considered where the calibration data are split into subsets with good linear relationship between y and x within each group. Different strategies for good prediction in this case are proposed. Modifications for collinear data are considered and a simple simulated data set is used for illustration.
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  • 28
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    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 5 (1991), S. 523-536 
    ISSN: 0886-9383
    Keywords: Measurement errors ; Method comparison ; Mean square successive difference ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Comparison of methods is a technique often used for investigation of systematic errors of measurement methods. As concerns the design and analysis of such comparisons, much variety of opinion and practice exists. In one approach a few specimens are measured several times by different operators in different laboratories (reproducibility conditions) and in another approach several specimens are measured on one or a few occasions by one operator in the same laboratory (repeatability conditions). In this paper a model for the error structure of measurements is formulated and it is emphasized that one has to distinguish between two types of systematic errors: the first type depends only on the level of the measured quantity and the second type is specific for the separate specimens. On the basis of this model the information which can be obtained from the different designs of method comparisons is discussed. A new approach for the analysis of method comparisons with many specimens is also proposed.
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  • 29
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 30
    ISSN: 0887-3585
    Keywords: site-directed mutagenesis ; ATP and AMP binding sites ; tryptophan fluorescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Site-directed mutagenesis of key amino acids of adenylate kinase has been used to suggest a new model for the location of the AMP and ATP binding sites. Phe-86 and Tyr-133, which are in close contact with the inhibitor Ap5A according to previous crystallographic results, have been independently changed to tryptophan and other amino acids. The Phe-86→Trp mutant had a 3- to 6-fold change in the Km for ATP and a 44-fold increase in the Km for AMP with a simultaneous loss of AMP substrate inhibition. Thus Phe-86 is probably in close contact with bound AMP. The Tyr-133→Trp mutant showed no large effects on enzyme kinetics and suggests that the previous assignment of Ap5A occupying natural adenosine binding sites is probably incorrect. A temperature-sensitive Leu-107→Gln mutant showed a 6-fold decrease in the Km for ATP and no effect on AMP binding, suggesting that this amino acid is near the ATP binding site.Changes in the fluorescence of single tryptophan-containing mutant enzymes provided specific information about AMP and ATP binding. The fluorescence results are consistent with the kinetic studies, and also suggest that AMP substrate inhibition is caused by the formation of an abortive complex that prevents the release of product.
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  • 31
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 32
    ISSN: 0887-3585
    Keywords: glutathione reductase ; X-ray analysis ; molecular replacement ; sitedirected mutagenesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of glutathione reductase from Escherichia coli has been solved at 3 Å resolution using multiple isomorphous replacement, solvent flattening, and molecular replacement on the basis of the homologous (53% identical residues) and structurally well-established human enzyme. The structures of both enzyme species agree with each other in a global way; there is no domain rearrangement. In detail, clear structural differences can be observed. The structure analysis of the E. coli enzyme was tackled in order to understand sitedirected mutants, the most spectacular of which changed the cofactor specificity of this enzyme from NADP to NAD (Scrutton et al., 1990, Nature 343:38-43).
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  • 33
    ISSN: 0887-3585
    Keywords: protein crystals ; crystallography ; latency ; tissue plasminogen activator ; N-terminal ; methionine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystals of bacterially expressed plasminogen activator inhibitor (PAI-1) suitable for X-ray diffraction analysis have been obtained from 8% (w/v) PEG 1500, pH 8.25. The space group is P1, and the lattice constants are a = 82.17 Å, b = 47.82 Å, c = 62.89 Å, α = 90.00°, β = 106.90°, γ = 106.84°. The diffraction limit is 2.3 Å, and the unit cell contains two molecules of PAI-1. The crystals contain latent PAI-1 which can be partly reactivated by exposure to denaturants.
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  • 34
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: phospholipase A2 ; interfacial catalysis ; interfacial activation ; resonance energy transfer ; lipid-protein interaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Interfacial catalysis is a necessary consequence for all enzymes that act on amphipathic substrates with a strong tendency to form aggregates in aqueous dispersions. In such cases the catalytic event occurs at the interface of the aggregated substrate, the overall turnover at the interface is processive, and it is influenced the molecular organization and dynamics of the interface. Such enzymes can access the substrate only at the interface because the concentration of solitary monomers of the substrate in the aqueous phase is very low. Moreover, the microinterface between the bound enzyme and the organized substrate not only facilitates formation of the enzyme-substrate complex, but a longer residence time of the enzyme at the substrate interface also promotes high catalytic processivity.Binding of the enzyme to the substrate interface as an additional step in the overall catalytic turnover permits adaptation of the Michaelis-Menten formalism as a basis to account for the kinetics of interfacial catalysis. As shown for the action of phospholipase A2 on bilayer vesicles, binding equilibrium has two extreme kinetic consequences. During catalysis in the scooting mode the enzyme does not leave the surface of the vesicle to which it is bound. On the other hand, in the hopping mode the absorption and desorption steps are a part of the catalytic turnover.In this minireview we elaborate on the factors that control binding of pig pancreatic phospholipase A2 to the bilayer interface. Binding of PLA2 to the interface occurs through ionic interactions and is further promoted by hydrophobic interactions which probably occur along a face of the enzyme, with a hydrophobic collar and a ring of cationic residues, through which the catalytic site is accessible to substrate molecules in the bilayer. An enzyme molecule binds to the surface occupied by about 35 lipid molecules with an apparent dissociation constant of less than 0.1 pM for the enzyme on anionic vesicles compared to 10 mM on zwitterionic vesicles. Results at hand also show that aggregation or acylation of the protein is not required for the high affinity binding or catalytic interaction at the interface.
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  • 35
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: staggering hydrogen bonds ; tilt ; twist ; βαβ crossover connection ; random generation of initial structures ; energy minimization ; associated loop energy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The folding of a polypeptide into a parallel (α/β)8 barrel (which is also called a circularly permuted β8α8 barrel) has been investigated in terms of energy minimization. According to the arrangement of hydrogen bonds between two neighboring β-strands of the central barrel therein, such an α/β barrel structure can be folded into six different types: (1) left-tilted, left-handed crossover; (2) left-tilted, right-handed crossover; (3) nontilted, left-handed crossover; (4) nontilted, right-handed crossover; (5) right-tilted, left-handed crossover; and (6) right-tilted, right-handed crossover. Here “tilt” refers to the orientational relation of the β-strands to the axis of the central β-barrel, and “crossover” to the βαβ folding connection feature of the parallel β-barrel. It has been found that the right-tilted, right-handed crossover α/β barrel possesses much lower energy than the other five types of α/β barrels, elucidating why the observed α/β barrels in proteins always assume the form of right tilt and right-handed crossover connection. As observed, the β-strands in the energy-minimized right-tilted, right-handed crossover (α/β)8-barrel are of strong right-handed twist. The value of root-mean-square fits also indicates that the central barrel contained in the lowest energy (α/β)8 structure thus found coincides very well with the observed 8-stranded parallel β-barrel in triose phosphate isomerase (TIM). Furthermore, an energetic analysis has been made demonstrating why the right-tilt, right-handed crossover barrel is the most stable structure. Our calculations and analysis support the principle that it is possible to account for the main features of frequently occurring folding patterns in proteins by means of conformational energy calculations even for very complicated structures such as (α/β)8 barrels.
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  • 36
    ISSN: 0887-3585
    Keywords: TIM ; molecular dynamics refinement ; loop movement ; conformational change ; crystal contacts ; sleeping sickness ; suramine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Triosephosphate isomerase has an important loop near the active site which can exist in a “closed” and in an “open” conformation. Here we describe the structural properties of this “flexible” loop observed in two different structures of trypanosomal triosephosphate isomerase. Trypanosomal triosephosphate isomerase, crystallized in the presence of 2.4 M ammonium sulfate, packs as an asymmetric dimer of 54,000 Da in the crystallographic asymmetric unit. Due to different crystal contacts, peptide 167-180 (the flexible loop of subunit-1) is an open conformation, whereas in subunit-2, this peptide (residues 467-480) is in a closed conformation. In the closed conformation, a hydrogen bond exists between the tip of the loop and a well-defined sulfate ion which is bound to the active site of subunit-2. Such an active site sulfate is not present in subunit-1 due to crystal contacts. When the native (2.4 M ammonium sulfate) crystals are transferred to a sulfate-free mother liquor, the flexible loop of subunit-2 adopts the open conformation. From a closed starting model, this open conformation was discovered through molecular dynamics refinement without manual intervention, despite involving Cα shifts of up to 7 Å. The tip of the loop, residues 472, 473, 474, and 475, moves as a rigid body. Our analysis shows that in this crystal form the flexible loop of subunit-2 faces a solvent channel. Therefore the open and the closed conformations of this flexible loop are virtually unaffected by crystal contacts. The actual observed conformation depends only on the absence or presence of a suitable ligand in the active site.
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  • 37
    ISSN: 0887-3585
    Keywords: mutant hemoglobin ; cooperativity ; protein structure ; conformational change ; quaternary structure ; allosteric proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Carbonmonoxy hemoglobin Ypsilanti (β99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of αβ dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the “switch” region, and packing interactions in the “flexible joint” region, show noncovalent interactions characteristic of the α1β2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the β97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.
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  • 38
    ISSN: 0887-3585
    Keywords: cryocrystallography ; temperature factor ; serine protease structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure of a rat trypsin mutant [S195C] at a temperature of 120 K has been refined to a crystallographic R factor of 17.4% between 12.0 and 1.59 Å and is compared with the structure of the D102N mutant at 295 K. A reduction in the unit cell dimensions in going from room temperature to low temperature is accompanied by a decrease in molecular surface area and radius of gyration. The overall structure remains similar to that at room temperature. The attainable resolution appears to be improved due to the decrease in the fall off of intensities with resolution [reduction of the temperature factor]. This decreases the uncertainty in the atomic positions and allows the localization of more protein atoms and solvent molecules in the low temperature map. The largest differences between the two models occur at residues with higher than average temperature factors. Several features can be localized in the solvent region of the 120 K map that are not seen in the 295 K map. These include several more water molecules as well as an interstitial sulfate ion and two interstitial benzamidine molecules.
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  • 39
    ISSN: 0887-3585
    Keywords: collective motion ; hinge bending motion ; normal mode analysis ; anharmonic motion ; low-frequency motion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A method is presented to describe the internal motions of proteins obtained from molecular dynamics or Monte Carlo simulations as motions of normal mode variables. This method calculates normal mode variables by projecting trajectories of these simulations onto the axes of normal modes and expresses the trajectories as a linear combination of normal mode variables. This method is applied to the result of the molecular dynamics and the Monte Carlo simulations of human lysozyme. The motion of the lowest frequency mode extracted from the simulations represents the hinge bending motion very faithfully. Analysis of the obtained motions of the normal mode variables provides an explanation of the anharmonic aspects of protein dynamics as due first to the anharmonicity of the actual potential energy surface near a minimum and second to trans-minimum conformational changes.
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  • 40
    ISSN: 0887-3585
    Keywords: α-helix ; side chain-backbone hydrogen bonding ; helix dipole ; circular dichroism ; carboxypeptidase A ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recently, Presta and Rose proposed1 that a necessary condition for helix formation is the presence of residues at the N-and C-termini (called NTBs and CTBs) whose side chains can form hydrogen bonds with the initial four amides and the last four carbonyls of the helix, which otherwise lack intrahelical hydrogen bonding partners. We have tested this hypothesis by conformational analysis by circular dichroism (CD) of a synthetic peptide corresponding to a region (171-188) of the protein carboxypeptidase A; in the protein, residues 174 to 186 are helical and are flanked by NTBs and CTBs. Since helix formation in this peptide may also be stabilized by electrostatic interactions, we have compared the helical content of the native peptide with that of several modified peptides designed to enable dissection of different contributions to helix stability. As expected, helix dipole interactions appear to contribute substantially, but we conclude that hydrogen bonding interactions as proposed by Presta and Rose also stabilize helix formation. To assist in comparison of different peptides, we have introduced two concentration-independent CD parameters which are sensitive probes of helix formation.
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  • 41
    ISSN: 0887-3585
    Keywords: calbindin D9K ; pseudo-EF hand ; peptide synthesis ; peptide design ; calcium ; α-helix ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A series of 37-residue analogues of the pseudo-EF hand in bovine calbindin D9K has been synthesized by the solid phase method. In the presence of calcium an α-helical induction of up to 44% was observed for the peptide with the native sequence with a Kd for calcium binding of 0.35 mM. A number of amino acid substitutions have been carried out to study the packing of the two α-helices based on the crystal structure of the entire protein. Three strategies were employed: (1) replacement of the Leu residues, which in the crystal structure do not contribute to the hydrophobic interaction between the two helices, by Gln or Ala in order to control the orientation of the helix packing, (2) stabilization of the individual helix by introducing a Glu-…Lys+ salt bridge or by changing the N-terminal charge to compensate for the helix dipole moment, and (3) introduction of a disulfide bond between the two helices to help the packing of the helices.The mutants with the substitution of (Leu-30, Leu-32) to (Gln-30, Gln-32), (Gln-30, Ala-32), and (Ala-30, Ala-32) designed based on the strategy 1 do not show any affinity for calcium and have low α-helicity. The Leu-30 to Lys-30 mutant designed to form a salt bridge between the side chains of Glu-26 and Lys-30 has an apparent Kd for calcium of 6.8 mM. Kd of the N-terminal acetylated and succinylated mutants are 0.41 and 0.45 mM, respectively, and no increase in the α-helix content relative to that of the natural sequence peptide is observed. The disulfide containing mutants, namely Tyr-13, Leu-31 to Cys-31 and Tyr-13, Leu-31 to Cys-13, hCys-31, show apparent Kd values of 0.93 and 2.1 mM, respectively. The former mutant shows the highest α-helix content among the peptides studied in the presence and absence of calcium. While it is difficult to construct an isolated and rigid helix-loop-helix motif with peptides of this size, introduction of a disulfide bond proved to be effective for this purpose.
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  • 42
    ISSN: 0887-3585
    Keywords: secondary structure ; tertiary structure ; residue conservation ; sequence variability ; sequence profile ; folding units ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The database of known protein three-dimensional structures can be significantly increased by the use of sequence homology, based on the following observations. (1) The database of known sequences, currently at more than 12,000 proteins, is two orders of magnitude larger than the database of known structures. (2) The currently most powerful method of predicting protein structures is model building by homology. (3) Structural homology can be inferred from the level of sequence similarity. (4) The threshold of sequence similarity sufficient for structural homology depends strongly on the length of the alignment. Here, we first quantify the relation between sequence similarity, structure similarity, and alignment length by an exhaustive survey of alignments between proteins of known structure and report a homology threshold curve as a function of alignment length. We then produce a database of homology-derived secondary structure of proteins (HSSP) by aligning to each protein of known structure all sequences deemed homologous on the basis of the threshold curve. For each known protein structure, the derived database contains the aligned sequences, secondary structure, sequence variability, and sequence profile. Tertiary structures of the aligned sequences are implied, but not modeled explicity. The database effectively increases the number of known protein structures by a factor of five to more than 1800. The results may be useful in assessing the structural significance of matches in sequence database searches, in deriving preferences and patterns for structure prediction, in elucidating the structural role of conserved residues, and in modeling three-dimensional detail by homology.
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  • 43
    ISSN: 0887-3585
    Keywords: structure database ; amino acid properties ; early folding intermediates ; stable peptide structures ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Patterns in amino acid properties (polar, hydrophobic, etc.) that characterize secondary structure motifs are derived from a database containing 75 protein structures, with the aim of circumventing the limitations due to data base size so as to increase structure prediction score. Many such sequence-structure associations with high intrinsic predictive power are found, which turn out to be correct 78% of the time when applied individually to proteins outside the learning set. Based on these associations, a prediction method is developed, which reaches the socre of 62% on the 3 states α-helix, β-strand, and loop, without using additional constraints. Though this score is quite good compared to that of other available prediction methods, it is much lower than could be expected from the high intrinsic predictive power of the associations used. The reasons underlying this surprising result, which indicate that prediction score and intrinsic predictive power are only weakly coupled, are discussed. It is also shown that the size of the present database still seriously limits prediction scores, even when property patterns are used, and that higher scores are expected in large databases. Clues are provided on the relative influence of neglecting spatial interactions on prediction efficiency, suggesting that, in sufficiently large databases, predicted secondary structures would correspond to those formed early in the folding process. This hypothesis is tested by confronting present predictions with available experimental data on early protein folding intermediates and on small peptides that adopt a relatively stable conformation in water. Although admittedly there are still too few such data, results suggest that the hypothesis might be well founded.
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  • 44
    ISSN: 0887-3585
    Keywords: plasmid SSBs ; protein and DNA sequence ; single-stranded DNA binding proteins ; helix destabilizing proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The DNA and protein sequences of single-stranded DNA binding proteins (SSBs) encoded by the plP71a, plP231a, and R64 conjugative plasmids have been determined and compared to Escherichia coli SSB and the SSB encoded by F-plasmid. Although the amino acid sequences of all of these proteins are highly conserved within the NH2-terminal two-thirds of the protein, they diverge in the COOH-terminal third region. A number of amino acid residues which have previously been implicated as being either directly or indirectly involved in DNA binding are conserved in all of these SSBs. These residues include Trp-40, Trp-54, Trp-88, His-55, and Phe-60. On the basis of these sequence comparisons and DNA binding studies, a role for Tyr-70 in DNA binding is suggested for the first time. Although the COOH-terminal third of these proteins diverges more than their NH2-terminal regions, the COOH-terminal five amino acid residues of all five of these proteins are identical. In addition, all of these proteins share the characteristic property of having a protease resistant, NH2-terminal core and an acidic COOH-terminal region. Despite the high degree of sequence homology among the plasmid SSB proteins, the F-plasmid SSB appears unique in that it was the only SSB tested that neither bound well to poly(dA) nor was able to stimulate DNA polymerase III holoenzyme elongation rates. Poly [d(A-T)] melting studies suggest that at least three of the plasmid encoded SSBs are better helix-destabilizing proteins than is the E. coli SSB protein.
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  • 45
    ISSN: 0887-3585
    Keywords: pattern recognition ; sequence alignment ; algorithms ; amino acid sequences ; molecular sequence data ; proteins ; software ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Multiple sequence alignment can be a useful technique for studying molecular evolution, as well as for analyzing relationships between structure or function and primary sequence. We have developed for this purpose an interactive program, MACAW (Multiple Alignment Construction and Analysis Workbench), that allows the user to construct multiple alignments by locating, analyzing, editing, and combining “blocks” of aligned sequence segments. MACAW incorporates several novel features. (1) Regions of local similarity are located by a new search algorithm that avoids many of the limitations of previous techniques. (2) The statistical significance of blocks of similarity is evaluted using a recently developed mathematical theory. (3) Candidate blocks may be evaluated for potential inclusion in a multiple alignment using a variety of visualization tools. (4) A user interface permits each blocks to be edited by moving its boundaries or by eliminating particular segments, and blocks may be linked to form a composite multiple alignment. No completely automatic program is likely to deal effectively with all the complexities of the multiple alignment problem; by combining a powerful similarity search algorithm with flexible editing, analysis and display tools, MACAW allows the alignment strategy to be tailored to the problem at hand.
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  • 46
    ISSN: 0887-3585
    Keywords: drug-design ; ligand-binding ; hemagglutinin ; functional groups ; MCSS ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new method is proposed for determining energetically favorable positions and orientations for functional groups on the surface of proteins with known three-dimensional structure. From 1,000 to 5,000 copies of a functional group are randomly placed in the site and subjected to simultaneous energy minimization and/or quenched molecular dynamics. The resulting functionality maps of a protein receptor site, which can take account of its flexibility, can be used for the analysis of protein ligand interactions and rational drug design. Application of the method to the sialic acid binding site of the influenza coat protein, hemagglutinin, yields functional group minima that correspond with those of the ligand in a cocrystal structure.
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  • 47
    ISSN: 0887-3585
    Keywords: third generation cephalosporins ; cefotaxime resistance ; enzyme structure-function ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The SHV-type β-lactamase SHV-2A is related to SHV-1 by a Gly-238-Ser replacement. Strains carrying SHV-2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV-1 are sensitive to these drugs. We present a kinetic analysis of a SHV-1 and SHV-2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV-2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7-aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV-1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV-2A. These results indicate that the Ser-238-Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV-2A relative to SHV-1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser-238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV-2A. We propose that the Gly-238 to Ser-238 replacement in SHV-2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.
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  • 48
    ISSN: 0887-3585
    Keywords: protein structure ; amino acid sequence ; database ; macromolecules ; molecular modeling ; knowledge base ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A system is described that provides ways of integrating data on protein structure, sequence, and survey results, with molecular graphics and molecular mechanics software. Its major component is the relational database SESAM, presently implemented under the commercial package SYBASE. By desin, the database allows full integration - within the same data organization - of raw data on protein structure, sequence, ligands, and heterogroups, obtained from the Brookhaven Protein Databank, with pure sequence information available from other databanks such as SWISS-PROT. It contains in addition higher level descriptions of structural and topological properties, as well as survey results, obtained by executing specialized computer programs. Aside from the very useful attribute of closely combining structural and nonstructural information, other important features distinguish it from analogous systems developed elsewhere. It includes a molecular dictionary with complete description of geometric properties and energy parameters used in modeling and conformational energy calculations. Using this dictionary, structural data are validated by checking for localized inconsistencies in atomic coordinates, atomic symbols, chirality definitions, and flagging errors and incomplete entries. Because of both the dictionary and the validation procedures, SESAM can be readily interfaced with conventional molecular graphics and mechanics software packages, or with other specialized application programs. With the aid of appropriate interfaces, data access is sufficiently fast for SESAM to be interrogated interactively. Prototypes of user interfaces, as well as an interface with the molecular graphics package BRUGEL, are described and the power of the system is illustrated in applications such as homology-based protein modeling, computer-aided protein design, protein structure predictions, analysis of local structure motifs, and of relationships between protein sequence and structure.
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  • 49
    ISSN: 0887-3585
    Keywords: expert system ; prediction from amino acid sequences ; sorting of proteins ; gram-negative bacteria ; genome analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We have developed an expert system that makes use of various kinds of knowledge organized as “if-then” rules for predicting protein localization sites in Gram-negative bacteria, given the amino acid sequence information alone. We considered four localization sites: the cytoplasm, the inner (cytoplasmic) membrane, the periplasm, and the outer membrane. Most rules were derived from experimental observations. For example, the rule to recognize an inner membrane protein is the presence of either a hydrophobic stretch in the predicted mature protein or an uncleavable N-terminal signal sequence. Lipoproteins are first recognized by a consensus pattern and then assumed present at either the inner or outer membrane. These two possibilities are further discriminated by examining an acidic residue in the mature N-terminal portion. Furthermore, we found an empirical rule that periplasmic and outer membrane proteins were successfully discriminated by their different amino acid composition. Overall, our system could predict 83% of the localization sites of proteins in our database.
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  • 50
    ISSN: 0887-3585
    Keywords: normal mode analysis ; potential energy function ; Lys-15 ; Arg-17 residues ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A normal mode analysis of bovine pancreatic trypsin inhibitor is carried out by using a Urey-Bradley-Shimanouchi potential energy function. The density of vibrational states, the magnitudes, and time scales of the atomic fluctuations are compared with experimental and theoretical results obtained by the more commonly used potential energy functions. The atomic fluctuations of Lys-15 are subject to extensive considerations as this residue is buried in the trypsin specificity pocket. It is found that Arg-17 is likely to be of importance in order to understand the way BPTI binds on trypsin.
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  • 51
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
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  • 52
    ISSN: 0887-3585
    Keywords: heme enzyme ; Pseudomonas putida ; conformational flexibility ; protein dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The structure and internal motions of cytochrome P-450cam, a monooxygenase heme enzyme with 414 amino acid residues, with camphor bound at the active site have been evaluated on the basis of a 175-psec molecular dynamics simulation carried out at 300 K. All hydrogen atoms were explicitly modeled, and 204 crystallographic waters were included in the simulation. Based on an analysis of the time course of the trajectory versus potential energy, root mean square deviation, radius of gyration, and hydrogen bonding, the simulation was judged to be stable and representative of the average experimental structure. The averaged structural properties of the enzyme were evaluated from the final 135 psec of the simulation. The average atomic displacement from the X-ray structure was 1.39 Å for all heavy atoms and 1.17 Å for just C-α atoms. The average root-mean-square (rms) fluctuations of all heavy atoms and backbone atoms were 0.42 and 0.37 Å, respectively. The computed rms fluctuations were in reasonable agreement with the experimentally determined temperature factors. All 13 segments of α-helix and 5 segments of β-sheet were well preserved with the exception of the N-terminal half of halix F which alternated between an α-helix and a 310-helix. In addition there were in general only small variations in the relative orientation of adjacent α-helices. The rms fluctuations of the backbone dihedral angles in the secondary structure elements were almost uniformly smaller, with the fluctuation in α-helices and β-sheets, 31 and 10% less, respectively, than those in nonsecondary structure regions. The reported crystal structure contains kinks in both helices C and I. In the simulation, both of these regions showed high mobility and large deviations from their starting positions. Since the kink in the I helix is at the oxygen binding site, these motions may have mechanistic implications.
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  • 53
    ISSN: 0887-3585
    Keywords: parotid saliva ; enzyme ; crystals ; X-ray analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Nonglycosylated α-amylase, a major component of human parotid saliva, has been crystallized by the vapor diffusion technique using 2-methyl-2,4-pentanediol as the precipitant in the presence of CaCl2 at pH 9.0. The crystals are orthorhombic, space group P212121 with unit cell dimensions of a = 53.3, b = 75.8, and c = 138.1 Å. The asymmetric unit contains one amylase molecule. The solvent content is 54%. The crystals are stable to X-rays and diffract up to 2.8 Å and appear to be suitable for X-ray diffraction studies.
    Additional Material: 2 Ill.
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  • 54
    ISSN: 0887-3585
    Keywords: protein crystals ; X-ray crystallography ; catechol O-methyltransferase ; protein structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Rat catechol O-methyltransferase cDNA was introduced into an E. coli expression vector pKEX14, which utilizes the inducible T7 promoter. Active and soluble recombinant catechol O-methyltransferase was produced in bacteria and purified to electrophoretic homogeneity by chromatographic procedures. The purified enzyme has been crystallized by the method of vapor diffusion using polyethylene glycol as precipitant. The space group is P3121 or P3221 with a = b = 51.3 Å and c = 168.5 Å and one molecule in the asymmetric unit. The crystals diffract beyond 3.2 Å and are suitable for three-dimensional X-ray structure determination.
    Additional Material: 3 Ill.
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  • 55
    ISSN: 0887-3585
    Keywords: antigen-antibody recognition ; protease-inhibitor complexes ; simulated annealing ; energy refinement ; docking algorithm ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Antibody-lysozyme and protease-inhibitor complexes are reconstituted by docking lysozyme as a rigid body onto the combining site of the antibodies and the inhibitors onto the active site of the proteases. Simplified protein models with one sphere per residue are subjected to simulated annealing using a crude energy function where the attractive component is proportional to the interface area. The procedure finds clusters of orientations in which a steric fit between the two protein components is achieved over a large contact surface. With five out of six complexes, the native structure of the complexes determined by X-ray crystallography is among those retained. Docked complexes are then subjected to conformational energy refinement with full atomic detail. With Fab HyHEL 5 and lysozyme, a native-like complex has the lowest refined energy. It can also be retrieved when starting with the X-ray structure of free lysozyme. However, some non-native complexes cannot be rejected: they form large interfaces, have a large number of H-bonds, and few unpaired polar groups. While these are necessary features of protein-protein recognition, they are not sufficient in determining specificity.
    Additional Material: 5 Ill.
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  • 56
    ISSN: 0887-3585
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The design of molecules to bind specifically to protein receptors has long been a goal of computer-assisted molecular design. Given detailed structural knowledge of the target receptor, it should be possible to construct a model of a potential ligand, by algorithmic connection of small molecular fragments, that will exhibit the desired structural and electrostatic complementarity with the receptor. However, progress in this area of receptorbased, de novo ligand design has been hampered by the complexity of the construction process, in which potentially huge numbers of structures must be considered. By limiting the scope of the structure-space examined to one particular class of ligands-namely, peptides and peptide-like compounds-the problem complexity has been reduced to the point that successful, de novo design is now possible. The methodology presented employs a large template set of amino acid conformations which are iteratively pieced together in a model of the target receptor. Each stage of ligand growth is evaluated according to a molecular mechanicsbased energy function, which considers van der Waals and coulombic interactions, internal strain energy of the lengtheining ligand, and desolvation of both ligand and receptor. The search space is managed by use of a data tree which is kept under control by pruning according to the energy evaluation. Ligands grown by this procedure are subjected to follow-up evaluation in which an approximate binding enthalpy is determined. This methodology has proven useful as a precise model-builder and has also shown the ability to design bioactive ligands.
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  • 57
    ISSN: 0887-624X
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The synthesis of poly(2,6-dimethyl-1,4-phenylene oxide) with one 2,6-dimethylphenol chain end (PPO-OH) and with well-defined molecular weight by phase transfer catalyzed polymerization of 4-bromo-2,6-dimethylphenol (20) in the presence of either 2,4,6-trimethylphenol (1) or 4-t-butyl-2,6-dimethylphenol (1′) as chain initiators is described. The range of controllable molecular weights and the mechanism of molecular weight control are discussed based on the differences between the reactivities of 20, 1, and 1′ and of the corresponding reactive species. The PPO-OH synthesized from 20/1′ has structural units derived from 1′ attached only at the chain end. PPO-OH synthesized from 20/1 contains structural units derived from 1 both internally and at the chain ends. Structural units derived from side reactions were identified by 1H-NMR spectroscopy. A reaction mechanism is proposed to account for their formation.
    Additional Material: 16 Ill.
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  • 58
    ISSN: 0887-624X
    Keywords: Chemistry ; Polymer and Materials Science
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Amorphous celluloses were prepared by regeneration of cellulose from its solutions in the SO2-diethylamine-dimethylsulfoxide (SO2-DEA-DMSO) solvent system, and other selected SO2-amine-organic solvents. The celluloses were amorphous whether regenerated in water or in alcohols or other organic solvents; in this respect the observations differ from all prior experience in the regeneration of cellulose. These celluloses retain their amorphous character even after extended soaking in water at room temperature. Viscosity measurements have shown that little or no depolymerization occurs during the dissolution, regeneration, and drying processes. Thus the procedures allow the preparation of amorphous celluloses of a wide range of molecular weights for use to model the behavior of amorphous domains in fibrous celluloses. The unusual stability of the amorphous cellulose structures prepared by these procedures is attributed to very rapid decomposition of the SO2-amine complex with the cellulosic hydroxyl groups which is believed to occur in the solvated state. The rate of decomposition of this complex appears to be sufficiently high so that the cellulose chains aggregate in an amorphous state before they have any opportunity to realign into crystalline domains.
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