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K A Taylor

Publications and source records attributed to K A Taylor.

At least 55 records · Page 3Linked to original sources
PubMed1993

Crossbridges in the complete unit cell of rigor insect flight muscle imaged by three-dimensional reconstruction from oblique sections.

We have computed two types of 3-D reconstructions from single images of oblique transverse sections through rigor insect flight muscle (IFM) that permit simultaneous examination of all myosin crossbridges within the unit cell. One type, crystallographic serial section reconstruction (CSSR), utilizes primarily real space image manipulations of the periodic crossbridge lattice to obtain a 3-D reconstruction from a single image. The CSSRs, which do not average successive unit cells along the filament axis, reveal variations in the rigor double chevrons within the 116 nm long axial repeat and in particular show that specific crossbridges are absent. CSSRs establish that in rigor, the 116 nm period contains nine 12.9 nm repeats of attached crossbridges rather than the eight 14.5 nm repeats of myosin head origins observed in the relaxed state. This indicates that dominance of the actin repeat on myosin head form enforces axial and azimuthal changes on the crossbridge origins on the thick filament. The second type, superlattice reconstruction (SLR), is carried out entirely in Fourier space and produces an averaged reconstruction with the symmetry of the unit cell enforced. SLRs measure the 3-D transform of the complete unit cell, permitting direct comparison with X-ray diagrams from native muscle. Averaging several SLRs together has produced the highest resolution reconstruction of IFM to date. Oblique section reconstructions made by both methods confirm in greater detail the presence of two-headed lead crossbridges and single-headed rear crossbridges implying heads with differing angles and conformation. Reduced twist in the thin filament coincident with the lead crossbridge is also apparent. We have modeled several interpretations of this reduced twist in terms of structural changes in both myosin and actin at the lead bridge. In addition, these 3-D images resolve a feature just Z-ward of the rear crossbridge where antibody labeling has identified part of the large troponin complex of IFM.

Actins↗
PubMed1993

Projection image of smooth muscle alpha-actinin from two-dimensional crystals formed on positively charged lipid layers.

Two-dimensional crystalline arrays of chicken gizzard alpha-actinin have been formed on positively charged lipid layers. This is the first reported crystallization of alpha-actinin. The crystals have unit cell dimensions of a = 248 A, b = 194 A, y = 106 degrees and contain two alpha-actinin molecules. The two-sided group is P21. Projection images obtained from electron micrographs of negatively stained crystals have been calculated to a resolution of 25 A. These images reveal a complex substructure. The molecule in projection is 340 A in length and has 12 density peaks that probably correspond to protein domains. A pair of peaks is found at each end of the molecule, these probably correspond to the actin binding region. Eight peaks are observed in the central, rod-shaped region, these may correspond to the spectrin-like repeats predicted from the amino acid sequence. However, these eight central peaks are not arranged in four pairs but, instead, consist of three central pairs flanked at either end by a single peak, which appears larger and denser in projection than the three central pairs. The individual alpha-actinin molecules in projection lack 2-fold symmetry suggesting that either smooth muscle alpha-actinin lacks a molecular 2-fold symmetry axis or that the molecular 2-fold is not parallel with the crystallographic 2-fold axis. The ends of the molecule have different appearance in projection, suggesting that the molecule is twisted about the long axis. A hypothesis is proposed to explain the variations in molecular length and Ca2+ sensitivity between alpha-actinin isoforms.

Actinin↗
PubMed1993

Comparison of two ribosomal DNA-based methods for differentiating members of the Anopheles gambiae complex (Diptera: Culicidae).

Two DNA-based methods, the restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR), were used to identify mosquitoes of the Anopheles gambiae Giles complex collected in Kenya. Field-collected specimens of An. gambiae, An. arabiensis Patton, and An. merus Donity were tested. From a sample of 208 mosquitoes, 181 (87%) were identified by the RFLP method and 205 (99%) were identified by the PCR method. There was complete concordance between the two methods with regard to species identification. PCR assays were simpler, faster, and more reliable than RFLP assays.

Animals↗
PubMed1993

A standard procedure for measuring pellet hardness of rodent diets.

A Chatillon Model TCM-200 test stand with exchangeable flat horizontal or concave receptacle bases and a DFI-200 gauge load cell with multiple types of upper exchangeable test jaws (large round-flat, medium round-flat, chisel, bullet, and cone-shaped) were compared by using preautoclaved and autoclaved NIH-31 rodent diet pellets to determine which type of hardness testing system would give the most accurate and reproducible results for measuring pellet hardness. The type and size of the contact area of the upper jaws significantly affected the force required to break the pellets. Significant differences were observed between the flat-horizontal and concave receptacle bases in the force required to break the pellets when using the two round-flat upper jaws. In contrast, similar results were obtained with both bases when the bullet, chisel, or cone-shaped upper jaws were used. Autoclaved pellets were 69.4% (range, 49 to 94%) harder than preautoclaved pellets. These results suggest that different testing systems can be used for measuring pellet hardness and that a standard procedure must be used in order to compare pellet hardness results between different testing laboratories. It was concluded that the flat-horizontal base and the larger round-flat end upper jaw gave the most reproducible results for measuring pellet hardness.

Animal Feed↗
PubMed1992

A colorimetric method for the quantitation of uronic acids and a specific assay for galacturonic acid.

A method of quantitating uronic acids and uronic acids from pectin in particular is described. The method uses carbazole in 80% sulfuric acid with borate ions added. The assay is carried out at 60 degrees C. This assay has some cross reactivity with aldose sugars and must be timed precisely. A further method that is specific for galacturonic acid is also described. This method uses concentrated sulfuric acid and carbazole only. Of the biological substances tested, only formaldehyde and glyceraldehyde showed a reactivity of more than 10% that of galacturonic acid on a weight to weight basis.

Boric Acids↗
PubMed1992

Sensitivity of a ribosomal RNA gene probe for identification of life stages of Anopheles arabiensis and An. gambiae (Diptera: Culicidae) using three storage methods.

Individual larvae, pupae, female adults, and adult body parts of Anopheles arabiensis Patton and An. gambiae Giles were stored for 1 mo either in isopropanol at room temperature, over a desiccant at room temperature, or at -70 degrees C. DNA was extracted, digested with EcoR1 restriction enzyme, subjected to electrophoresis in agarose gel, transferred to filters, then hybridized to a 32P-labeled rDNA probe. There was no difference among storage treatments in the proportion of correctly identified samples. First instars were not identifiable. Pupae and female adults were more likely to be identified than earlier life history stages. Nonetheless, the probe identified greater than 75% of second instars, 94% of third instars, and 74% of fourth instars. There were no differences between the species in the proportion of identifiable samples for any life history stage.

Animals↗
PubMed1992

Structural characterization of ordered arrays of sn-glycerol-3-phosphate acyltransferase from Escherichia coli.

Overproduction of the sn-glycerol-3-phosphate acyltransferase in Escherichia coli leads to incorporation of this integral membrane protein into ordered tubular arrays within the cell. Freeze-fracture-etch shadowing was performed on suspensions of partially purified tubules and whole bacteria. This procedure revealed the presence of ridges and grooves defining a set of long-pitch left-handed helical ridges. The long-pitch helices represented chains of acyltransferase dimers. Tubules observed within the cell were often closely packed, with an apparent alignment of grooves and ridges in adjacent tubules. Fracture planes passing through the tubules indicated the presence of a bilayer structure, with some portion of the enzyme being associated with the membrane. The major portion of the enzyme extended from the hydrophilic surface, forming a large globular structure that, in favorable views, displayed a central cavity facing the cytoplasm. Computer analysis of shadowed tubules revealed that the left-handed helices were six stranded, with a pitch of 1,050 A (105.0 nm) and a spacing of 75 A (7.5 nm) between acyltransferase dimers along the chains. Analysis of the predicted secondary structure failed to reveal obvious transmembrane segments, suggesting that very little of the protein was inserted into the bilayer.

Cytoplasm↗
PubMed1991

Effects of solutes on the formation of crystalline sheets of the Ca(2+)-ATPase in detergent-solubilized sarcoplasmic reticulum.

The Ca(2+)-ATPase crystals formed in detergent solubilized sarcoplasmic reticulum (SR) at 2 degrees C in a crystallization medium of 0.1 M KCl, 10 mM K-Mops (pH 6.0), 3 mM MgCl2, 3 mM NaN3, 5 mM DTT, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 20% glycerol and 20 mM CaCl2 (J. Biol. Chem. 263, 5277 and 5287 (1988)) contain highly ordered sheets of ATPase molecules, that associate into large multilamellar stacks (greater than 100 layers). When the crystallization is performed in the same medium but in the presence of 40% glycerol at low temperature the stacking is reduced to 4-5 layers and the average diameter of the crystalline sheets is increased from less than 1 micron to 2-3 microns. Glycerol and low temperature presumably reduce stacking by interfering with the interactions between the hydrophilic headgroups of Ca(2+)-ATPase molecules in adjacent lamellae, while not affecting or promoting the ordering of ATPase molecules within the individual sheets. Electron diffraction patterns could be regularly obtained at 8 A and occasionally at 7 A resolution on crystals formed in 40% glycerol, either at 2 degrees C or at -70 degrees C. In the same media but in the absence of glycerol, polyethyleneglycol 1450, 3000 and 8000 (1-8%) induced the formation of ordered crystalline arrays containing 10-12 layers that were similar to those obtained in 40% glycerol. Replacement of 40% glycerol with 10-50% glucose or supplementation of the standard crystallization medium with polyethyleneglycol (PEG 3000 or 8000; 1, 2, 5 and 8%) had no beneficial effect on the order of crystalline arrays compared with media containing 40% glycerol.

Animals↗
PubMed1991

A protocol for 3D image reconstruction from a single image of an oblique section.

Oblique section 3D reconstruction can produce a 3D image of a sectioned crystal from a single electron micrograph. We describe here in detail a reconstruction protocol applicable to an electron micrograph of an oblique section through a 3D crystal. The protocol is described in six steps: (1) selection criteria for images, (2) preprocessing steps to correct for image defects, (3) determination of unit cell coordinates, (4) interpolation of strip images with correction for image distortions and crystal disorder, (5) production of a crystallographic serial section reconstruction, (6) correction for skewed sampling to produce an oblique section reconstruction. In addition, we explore Wiener filter deconvolution of the section thickness. We describe a method for determining the section thickness by comparing data from projections of the oblique section reconstruction with corresponding data from a thick longitudinal section. Several schemes for Wiener filter deconvolution are described that differ in the way information on the signal-to-noise ratio is used in the filter.

Animals↗
PubMed1991

Pulsed-field gel electrophoretic analysis of leptospiral DNA.

The genomic structures of spirochete species are not well characterized, and genetic studies on these organisms have been hampered by lack of a genetic exchange mechanism in these bacteria. In view of these observations, pulsed-field gel electrophoresis was used to examine the genomes of Leptospira species. Live cells, prepared in agarose plugs, were lysed in situ, and the DNA was analyzed under different electrophoretic conditions. Pulsed-field gel electrophoresis of DNA digested with infrequently cutting restriction enzymes showed that the genome of Leptospira interrogans serovar canicola is approximately 3.1 Mb, while that of the saprophytic L. biflexa serovar patoc I is 3.5 Mb. DNA forms of approximately 2,000 and 350 kb which were present in samples from L. interrogans serovars were not readily detected in nonpathogenic serovars. Three distinct populations, designated type alpha, beta, and gamma, of L. interrogans DNA molecules were further analyzed with two-dimensional gel electrophoresis. Evidence suggested that two of these DNA forms, type alpha and gamma, were linear structures. Pulsed-field gel electrophoresis has proven to be a valuable tool with which to size bacterial genomes and to take the first steps toward characterization of a form of leptospiral DNA which behaves as a linear molecule and which may be related to the virulence of L. interrogans.

DNA, Bacterial↗
PubMed1990

Computation of a three dimensional image of a periodic specimen from a single view of an oblique section.

We describe here a method for computing a three dimensional map of a periodic specimen from a single electron micrograph of an obliquely cut section. Neighbouring areas of such an image display successively the contents of the unit cell of the structure. The reconstruction procedure can be considered in two steps. The first step involves restacking of successive areas to produce an image akin to that produced by serial section reconstruction. The resolution normal to the section would, at this stage, be limited by the thickness of the section, since the micrograph represents a projection of the density in the section. However, because of the periodic nature of the specimen, the image contains redundant information, which can be used in an attempt to deconvolute the section thickness and thus produce improved resolution normal to the section. The computation can be carried out directly with the densities or more conveniently, particularly for three dimensional crystals, by using Fourier transforms. The approach, which is most powerful when the section is thin, is insensitive to the collapse of the section caused by electron irradiation. Striated muscle provides particularly suitable specimens for such analysis and we present, as examples, computed maps of the M-band of fish muscle and of insect flight muscle in rigor.

Animals↗
PubMed1990

Plasmodium falciparum infection rates in Anopheles gambiae, An. arabiensis, and An. funestus in western Kenya.

Mosquitoes collected monthly for 1 year from human habitations in the Kisumu area of western Kenya were identified by morphological characters as Anopheles gambiae Giles sensu lato (An. gambiae s.l.) or An. funestus. Of the mosquitoes collected, 7,244 (67%) of the An. gambiae s.l. and 8,511 (87%) of the An. funestus were tested by enzyme-linked immunosorbent assay (ELISA) for the presence of Plasmodium falciparum circumsporozoite (CS) protein. ELISA positivity rates were 8.2% for An. gambiae s.l. and 6.1% for An. funestus. Both An. gambiae and An. arabiensis were detected among 432 ELISA-positive and 668 ELISA-negative An. gambiae s.l. identified to species with a ribosomal DNA probe. The species-specific infection rates were calculated to be 9.6% for An. gambiae and 0.4% for An. arabiensis. These results confirm that An. gambiae and An. funestus are the primary malaria vectors in western Kenya and that An. arabiensis is a relatively minor vector.

Animals↗
PubMed1990

The effect of oxygen and carbon dioxide on the development of the free-living stages of Strongyloides ratti in axenic culture.

The effect of oxygen and carbon dioxide concentration on the development of the free-living stages of Strongyloides ratti in axenic culture was examined. Hatching of S. ratti eggs was inhibited at very low oxygen levels. Development of all organisms was inhibited in medium gassed with less than 4% O2 in 0.03% CO2 with the remainder N2. Between 8 and 21% O2 there was no significant difference in the percentage developing directly to filariform larvae, free-living females, or free-living males. Cultures gassed with CO2 concentrations greater than 1% in 21% O2 with the remainder N2 manifested inhibited development to filariform larvae, whereas concentrations between 5 and 7.5% enhanced development to free-living females. There was a positive effect on increasing the CO2 concentration from 0.03% to 5% on development to filariform larvae and male worms at levels of 1% and 2% oxygen in nitrogen, respectively. Direction of development, either directly to the filariform larva or to the free-living female, was influenced by CO2 and O2 concentrations encountered by the egg or the newly hatched larva of the parasitic female in axenic culture. The axenic culture system permits an important experimental approach to the study of factors modulating the differentiation of the free-living stages of Strongyloides.

Animals↗
PubMed1989

Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer.

We have obtained detailed three-dimensional images of in situ cross-bridge structure in insect flight muscle by electron microscopy of multiple tilt views of single filament layers in ultrathin sections, supplemented with data from thick sections. In this report, we describe the images obtained of the myac layer, a 25-nm longitudinal section containing a single layer of alternating myosin and actin filaments. The reconstruction reveals averaged rigor cross-bridges that clearly separate into two classes constituting lead and rear chevrons within each 38.7-nm axial repeat. These two classes differ in tilt angle, size and shape, density, and slew. This new reconstruction confirms our earlier interpretation of the lead bridge as a two-headed cross-bridge and the rear bridge as a single-headed cross-bridge. The importance of complementing tilt series with additional projections outside the goniometer tilt range is demonstrated by comparison with our earlier myac layer reconstruction. Incorporation of this additional data reveals new details of rigor cross-bridge structure in situ which include clear delineation of (a) a triangular shape for the lead bridge, (b) a smaller size for the rear bridge, and (c) density continuity across the thin filament in the lead bridge. Within actin's regular 38.7-nm helical repeat, local twist variations in the thin filament that correlate with the two cross-bridge classes persist in this new reconstruction. These observations show that in situ rigor cross-bridges are not uniform, and suggest three different myosin head conformations in rigor.

Actins↗
PubMed1989

Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer.

The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice.

Actins↗
PubMed1988

Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum.

Crystalline arrays of Ca2+-ATPase molecules develop in detergent-solubilized sarcoplasmic reticulum during incubation for several weeks at 2 degrees C under nitrogen in a medium of 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Electron microscopy of sectioned, negatively stained, freeze-fractured, and frozen-hydrated Ca2+-ATPase crystals indicates that they consist of stacked lamellar arrays of Ca2+-ATPase molecules. Prominent periodicities of ATPase molecules within the lamellae arise from a centered rectangular lattice of dimensions 164 x 55.5 A. The association of lamellae into three-dimensional stacks is assumed to involve interactions between the exposed hydrophilic headgroups of ATPase molecules, that is promoted by glycerol and 20 mM Ca2+. Similar Ca2+-induced crystals were observed with purified or purified and delipidated Ca2+-ATPase preparations at lower detergent/protein ratios. Cross-linking of Ca2+-ATPase crystals with glutaraldehyde protects the structure against conditions such as low Ca2+, high pH, elevated temperature, SH group reagents, high concentration of detergents, and removal of phospholipids by extraction with organic solvents that disrupt unfixed preparations.

Animals↗
PubMed1988

Absence of detectable chromosomal and molecular abnormalities in monozygotic twins discordant for the Wiedemann-Beckwith syndrome.

Monozygotic twins discordant for the Wiedemann-Beckwith syndrome (WBS) were studied by cytogenetic and molecular methods to determine if a genetic lesion could be detected in the affected child. Probes known to be localized on the short arm of chromosome 11 and a low copy-repetitive probe were used. No genetic lesions could be ascertained in normal or affected tissue obtained from the WBS twin.

Beckwith-Wiedemann Syndrome↗