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Biomedical subjects

K A Taylor

Publications and source records attributed to K A Taylor.

At least 37 records · Page 2Linked to original sources
PubMed1996

Electron tomography of insect flight muscle in rigor and AMPPNP at 23 degrees C.

Treatment of rigor fibers of insect flight muscle (IFM) with AMPPNP at 23 degrees C causes a 70% drop in tension with little change in stiffness. In order to visualize the changes in crossbridge conformation and distribution that give rise to the mechanical response, we have produced three-dimensional reconstructions by tomography of both rigor and AMPPNP-treated muscle that do not average the repeating motifs of crossbridges, and thereby retain information on variability of crossbridge structure and distribution. Tomograms can be averaged when display of only the regular features is wanted. Tomograms of rigor IFM show double-headed lead and single-headed rear crossbridges. Tomograms of IFM treated with AMPPNP at 23 degrees C reveal many double-headed and some single-headed "lead" bridges but few crossbridges corresponding to the rear bridges of rigor. Instead, new non-rigor forms of variably angled crossbridges are found bound to actin sites not labeled with myosin heads in rigor. This indicates that the rear bridges of rigor have redistributed during the transition from rigor to the AMPPNP state, which could explain the maintenance of rigor stiffness despite the loss of tension. Comparison of in situ crossbridges in tomograms of rigor with atomic model of acto-S1, the complex formed by myosin subfragment 1 and actin, reveals that the regulatory domain of S1 would require significant bending and realignment to fit into both types of rigor crossbridges. The modifications are particularly significant for the rear bridges and suggest that differential strain in the regulatory domain of rear bridges may be the basis for their detachment and redistribution upon binding AMPPNP. Similar comparison using lead-type crossbridges in AMPPNP reveals departures from the rigor acto-S1 atomic model that include azimuthal straightening and a slight M-ward bending in the regulatory domain. Both the motor and regulatory domains of the new non-rigor crossbridges differ from those in the atomic model of acto-S1. A new crossbridge motif identified in AMPPNP-treated muscle consists of paired rigor-like and non-rigor crossbridges and suggests possible transitions in the myosin working stroke.

Adenosine Triphosphate↗
PubMed1996

Three-dimensional structure of nucleotide-bearing crossbridges in situ: oblique section reconstruction of insect flight muscle in AMPPNP at 23 degrees C.

We have explored the three-dimensional structure of myosin crossbridges in situ in order to define the structural changes that occur when nucleotide binds to the myosin motor. When AMPPNP binds to rigor insect flight muscle, each half sarcomere lengthens by approximately 2.0 nm and tension is reduced by approximately 70% without a reduction in stiffness, suggesting partial reversal of the power stroke. We have obtained averaged oblique section three-dimensional reconstructions of mechanically monitored insect flight muscle in AMPPNP that permit simultaneous examination of all myosin crossbridges within the unit cell and direct comparison of calculated transforms with X-ray diagrams of the native fibers. Transforms calculated from the oblique section reconstruction of AMPPNP insect flight muscle at 23 degrees C show good agreement with native X-ray diagrams, suggesting that the average crossbridge forms in the reconstruction reflect the native structure. In contrast to the rigor lead and rear crossbridges in the double chevrons, the averaged reconstruction of AMPPNP fibers show only one crossbridge class, in the position of the rigor lead bridge. The portion of the crossbridge close to the thick filament appears broader than in rigor, and shows a small 0.5 to 1.0 nm M-ward shift of the regulatory domain region of myosin. In transverse view, AMPPNP "lead" crossbridges are less azimuthally bent than rigor. Fitting the atomic model of actomyosin subfragment 1 to the averaged crossbridges shows that the detectable differences between rigor bridges and between rigor and AMPPNP bridges occur in the alignment and angles of the regulatory domains and suggests that rear bridges are more strained than lead crossbridges. The apparent absence of rear bridges in AMPPNP in averaged reconstructions indicates detachment of a number of force-bearing bridges, which conflicts with the maintained stiffness of the fibers used for the reconstruction. This conflict may be explained if rigor rear bridges become distributed irregularly over more actin sites in AMPPNP, so that their average density is too low to appear in the averaged reconstructions. The reconstructions indicate that in insect flight muscle the response of in situ rigor crossbridges to AMPPNP binding is not uniform. Lead bridges persist but have altered structure in the light chain domain, whereas rear bridges detach and possibly redistribute. Shape changes in attached myosin heads within the myofibrillar lattice are in the appropriate direction and of the appropriate magnitude needed to explain the sarcomere lengthening. This could be a direct response to nucleotide binding, a passive response to rear bridge detachment, or a combination of both.

Adenosine Triphosphate↗
PubMed1996

Bacterial cell division protein FtsZ assembles into protofilament sheets and minirings, structural homologs of tubulin polymers.

The bacterial cell division protein FtsZ is a homolog of tubulin, but it has not been determined whether FtsZ polymers are structurally related to the microtubule lattice. In the present study, we have obtained high-resolution electron micrographs of two FtsZ polymers that show remarkable similarity to tubulin polymers. The first is a two-dimensional sheet of protofilaments with a lattice very similar to that of the microtubule wall. The second is a miniring, consisting of a single protofilament in a sharply curved, planar conformation. FtsZ minirings are very similar to tubulin rings that are formed upon disassembly of microtubules but are about half the diameter. This suggests that the curved conformation occurs at every FtsZ subunit, but in tubulin rings the conformation occurs at either beta- or alpha-tubulin subunits but not both. We conclude that the functional polymer of FtsZ in bacterial cell division is a long thin sheet of protofilaments. There is sufficient FtsZ in Escherichia coli to form a protofilament that encircles the cell 20 times. The similarity of polymers formed by FtsZ and tubulin implies that the protofilament sheet is an ancient cytoskeletal system, originally functioning in bacterial cell division and later modified to make microtubules.

Animals↗
PubMed1996

Trypanosoma congolense: B-lymphocyte responses differ between trypanotolerant and trypanosusceptible cattle.

Trypanosomiasis is a serious constraint to livestock production in sub-Saharan Africa. Some breeds of cattle are genetically more resistant to the pathogenic effects of trypanosome infection. We measured B-cell activation and the quantity and isotype of antibody produced at the cellular level in six trypanotolerant N'Dama and five trypanosusceptible Boran cattle. The frequencies of spleen cells secreting total and parasite-specific IgM and IgG were measured prior to and 16, 28, and 35 days after a primary challenge with Trypanosoma congolense. Boran cattle had higher frequencies of splenic cells secreting IgM specific for trypanosome-derived variable surface glycoprotein (VSG), cysteine protease (congopain, CP), and heat shock protein (hsp 70/BiP) and the nonparasite antigen, ovalbumin, than did N'Dama cattle. In contrast, the number of VSG-specific IgG-secreting cells was significantly greater in N'Dama than in Boran cattle. During infection, low titers of anti-VSG IgM were detected transiently in the serum of all animals. However, N'Dama had significantly more VSG-specific IgG in blood than Boran during infection. The peripheral blood mononuclear cell population of N'Dama cattle contained a higher percentage of surface IgM-positive B-cells prior to and throughout infection than were found in the blood of Boran. In addition, during infection N'Dama cattle had more circulating lymphocytes that could be activated in vitro to undergo differentiation into IgM- and IgG-secreting cells. These findings demonstrate differences in the frequency of trypanosome-specific antibody-secreting cells in the spleen and in the activation state of B-cells in the blood between N'Dama and Boran cattle during a primary infection with T. congolense.

Analysis of Variance↗
PubMed1996

Trypanosoma congolense: a comparison of T-cell-mediated responses in lymph nodes of trypanotolerant and trypanosusceptible cattle during primary infection.

A comparison of T-cell-mediated immune responses in trypanotolerant N'Dama and susceptible Boran cattle during primary infection with tsetse-transmitted Trypanosoma congolense was conducted to assess whether different patterns of T-cell activation occurred during trypanosome infection. Proliferation and IFN-gamma synthesis in response to trypanosome antigens and to the mitogen Con A were measured in LNC before infection and 10 and 35 days postinfection. Phenotypic analysis of LNC was also carried out. No significant differences in the in vitro proliferation of LNC to VSG, to hsp70/BiP, or to Con A were detected between the breeds. In contrast, IFN-gamma production in response to Con A was higher in Boran cattle at 35 days p.i. A reduction in the number of CD2+ and CD4+ T-cells and an increase in the percentage of B-cells, CD8+ T-cells, and gamma delta T-cells during infection in both N'Dama and Boran was revealed by cytofluorimetric analysis of lymph node cells.

Animals↗
PubMed1996

Exercise training mode affects the hemodynamic responses to lower body negative pressure in women.

To determine if different exercise modes used to improve cardiovascular fitness result in differing cardiovascular responses to lower body negative pressure (LBNP) in exercise-trained women, seven chronically exercising female runners (RUN) and 11 swimmers (SWIM) of similar fitness levels maximal oxygen uptake, [VO(2max), mean (SEM) = 50 (2) and 45 (2) ml*kg(-1)*min(-1), respectively; P > 0.05] underwent serial exposures to LBNP at pressures of 0, -1.3, -2.7 and -5.3 kPa (referenced to ambient barometric pressure). Forearm vascular resistance (venous occlusion plethysmography) increased with LBNP but did not differ between groups at any level of LBNP. At 0 and - 1.3 kPa, the total peripheral resistance index (TPRI; impedance cardiography) was significantly (P <0.05) higher in RUN than SWIM [1.118(0.028) vs 0.787 (0.040) at 0 kPa and 1.245 (0.100) vs 0.840 (0.040) 2 kPa*l* min(-1)*m(-2) at -1.3kPa]. At an LBNP of -2.7kPa, stroke index (SI) was significantly higher in SWIM than RUN [57.8(4.6) vs 41.9 (4.0) ml*beat(-1)*m(-2)] while TPRI remained greater in RUN than SWIM. At -5.3 kPa, SWIM exhibited a higher cardiac index [3.232 (0.209) vs 2.447 (0.189) l*min(-1)*m(-2)] and SI [49.4 (4.4) vs 31.0 (4.5) ml *beat(-1)*m(-2)] but reduced heart rate [71(3) vs 83(5) beats . min(-1)] and TPRI [0.968 (0.043) vs 1.655 (0.128) kPa*1*min(-1)* m(-2)]. Mean arterial pressure declined significantly at an LBNP of -5.3 kPa in both groups; pulse pressure was lower (P <0.05) in RUN than SWIM at LBNP values of -2.7 and -5.3 kPa. These data suggest that: (1) female runners experience a greater increase in systemic vasoconstriction even though female swimmers can better maintain their cardiac index at high levels of LBNP, and (2) training mode appears to affect the pulse pressure responses to LBNP in exercise-trained women.

Adult↗
PubMed1996

Plasmodium falciparum circumsporozoite vaccine immunogenicity and efficacy trial with natural challenge quantitation in an area of endemic human malaria of Kenya.

It has been hypothesized that antibody induced by Plasmodium falciparum circumsporozoite protein vaccine would be effective against endemic human malaria. In a malaria endemic region of Kenya, 76 volunteers, in 38 pairs sleeping adjacently, were immunized with subunit circumsporozoite protein Asn-Ala-Asn-Pro tetrapeptide repeat-pseudomonas toxin A, or hepatitis B vaccine. After quinine and doxcycycline, volunteers were followed for illness daily, parasitemia weekly, antibody, T-lymphocyte responses, and treated if indicated. Anopheles mosquitoes resting in houses were collected, and tested for P. falciparum antigen, or dissected for sporozoites and tested for blood meal ABO type and P. falciparum antigen. Vaccine was safe, with side-effects similar in both groups, and immunogenic, engendering IgG antibody as high as 600 micrograms ml-1, but did not increase the proportion of volunteers with T-lymphocyte responses. Estimation of P. falciparum challenge averaged 0.194 potentially infective Anopheles bites/volunteer/ day. Mosquito blood meals showed no difference in biting intensity between vaccine and control groups. Both groups had similar malaria-free survival curves, cumulative positive blood slides, cumulative parasites mm-3, and numbers of parasites mm-3 on first positive blood slide, during three post-vaccination observation periods. Every volunteer had P. falciparum parastemia at least once. Vaccinees had 82% and controls 89% incidences of symptomatic parasitemia (P = 0.514, efficacy 9%, statistical power 95% probability of efficacy < 50%). Vaccine-induced anti-sporozoite antibody was not protective in this study. Within designed statistical precisions the present study is in agreement with efficacy studies in Colombia, Venezuela and Tanzania.

Animals↗
PubMed1996

Three-dimensional distortion correction applied to tomographic reconstructions of sectioned crystals.

Unaveraged tomograms of embedded and sectioned insect flight muscle show not only structural variation within the individual unit cells but also defects in the filament lattice such as lack of straightness of the filaments and wrinkling of the sections. Adaptation of conventional 2D crystal image processing methods usually average out these structural variations and lattice defects thereby reducing the resolution and information content of the 3D reconstructions. We present here a computational method to correct the reconstructed 3D object for these distortions to make it suitable for further analysis. The correction is an extension of conventional 2D cross-correlation techniques applied to a 3D image. These procedures determine the unit cell positions of the distorted crystal and use a spline-fitting approach to calculate a mapping function from the observed positions that is subsequently used to reinterpolate the image onto a regular grid. The resulting reinterpolated image is corrected for both filament straightening as well as section wrinkling.

Animals↗
PubMed1995

Intragenic matrix attachment and DNA-protein interactions in the human X-linked Hprt gene.

To investigate the possible contribution of intragenic differentially methylated cytosines to X-linked gene expression, we examined DNA-protein interactions in a region in intron 3 of the human hypoxanthine phosphoribosyltransferase gene which contains at least one HpaII site methylated specifically on the active X. In vitro DNase I footprinting experiments using unmethylated DNA and HeLa nuclear extract identified three footprints (I-III). Footprints I and III flank an Alu repeat containing the HpaII site(s), one of which is contained within footprint II. Although methylation of the HpaII site had no effect on footprint II binding interactions, methylation of nearby CpGs substantially reduced the formation of three of the specific DNA-protein complexes binding to footprint II in mobility shift assays. Additionally, an A+T rich region immediately 5' to the HpaII-containing Alu repeat was found to bind specifically to nuclear matrices in vitro. We suggest that differential methylation of CpGs may affect the binding of regulatory proteins in vivo, and that interactions between the footprint proteins and those binding to the matrix attachment region may be involved in controlling X-linked Hprt expression.

Base Sequence↗
PubMed1995

Trypanosoma congolense: tissue distribution of long-term T- and B-cell responses in cattle.

Memory T- and B-cell responses to trypanosome antigens were measured in peripheral blood mononuclear cells, spleen and lymph node cells obtained from four trypanotolerant N'Dama cattle which had been exposed to six experimental infections with Trypanosoma congolense. These cattle were treated with trypanocidal drugs following each infection and had remained aparasitemic for 3 years prior to this study. The antigens used were whole trypanosome lysate, variable surface glycoprotein, a 33-kDa cysteine protease (congopain) and a 70-kDa heat-shock protein. As parameters of T-cell-mediated immunity, we measured T-cell proliferation and IFN-gamma production. Lymph node cells, spleen cells and peripheral blood mononuclear cells all proliferated to a mitogenic stimulus (concanavalin A) but only lymph node cells responded to trypanosome antigens. Similarly, IFN-gamma was produced by both lymph node and spleen cells stimulated with concanavalin A but only by lymph node cells stimulated with variable surface glycoprotein and whole trypanosome lysate. T. congolense-specific antibodies were detected in sera and in supernatants of cultured lymph node and spleen cells after in vitro stimulation with lipopolysaccharide and recombinant bovine interleukin-2. In conclusion, we have demonstrated that memory T- and B-cell responses are detectable in various lymphoid organs in cattle 3 years following infection and treatment with T. congolense.

Animals↗
PubMed1994

Similarity of three-dimensional microcrystals of detergent-solubilized (Na+,K+)-ATPase from pig kidney and Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum.

Image analysis has been used to compare the projection structure of three-dimensional crystals of (Na+,K+)-ATPase from pig kidney and the Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. These crystals, formed from detergent-solubilized protein in the presence of 20% glycerol and at a low detergent to protein ratio, crystallize in nearly identical unit cells in the space group C2. Average cell dimensions for the Ca(2+)-ATPase were a = 166.8 +/- 4.5 A, b = 57.7 +/- 4.4 A, gamma = 90 degrees while those for the (Na+,K+)-ATPase were a = 166.2 +/- 3.8A, b = 54.25 +/- 3.5A, gamma = 90 degrees. Their projected structures at the resolution of 25-A resolution are indistinguishable. Thus, the (Na+,K+)-ATPase crystals appear to contain only the alpha chain of the alpha beta heterodimer found in native membranes. We conclude from this that the three-dimensional structure of the alpha chain of the (Na+,K+)-ATPase is very similar to that of the Ca(2+)-ATPase despite their relatively weak overall sequence homology.

Animals↗
PubMed1994

In vitro activation and detection of antibody-secreting cells from Trypanosoma congolense-infected cattle.

B cells from the peripheral blood and spleen of Trypanosoma congolense-infected cattle and from the peripheral blood of an uninfected cohort were analysed for ability to secrete antibody and for expression of surface antigens before and after in vitro culture with interleukin-2, lipopolysaccharide and pokeweed mitogen. Antibody-secreting cells (ASC) were only detected in lymphocytes from peripheral blood after in vitro stimulation. The frequency of ASC was greater in cultures of lymphocytes from infected cattle than from the uninfected cohort. The frequency of ASC was positively correlated with the number of B cells expressing the transferring receptor but not with the expression of the CD5 antigen.

Animals↗
PubMed1994

3D reconstruction by combining data from sections cut oblique to different unit cell axes.

Oblique section reconstruction (OSR) can produce a 3D image from a single micrograph of a section through a 2D or 3D crystal. Resolution, however, is limited in a direction perpendicular to the section plane. When data from sections cut at different orientations are combined, the inherent resolution limitation for single sections can be overcome. In the extreme, combination of sections cut oblique to different principal planes of the unit cell can produce a 3D reconstruction with isotropic resolution. The merging of data from different sections requires determination of section thickness and orientation. A method for section thickness determination is described that is generally applicable event to space groups with little or no symmetry. In addition, an approach has been developed to determine rotational orientation within the hexagonal lattice of insect flight muscle. Finally, an iterative least-squares fitting method is applied to determine a 3D set of deconvoluted structure factors from the merged data.

Adenylyl Imidodiphosphate↗
PubMed1994

Oblique section 3-D reconstruction of relaxed insect flight muscle reveals the cross-bridge lattice in helical registration.

In this work we examined the arrangement of cross-bridges on the surface of myosin filaments in the A-band of Lethocerus flight muscle. Muscle fibers were fixed using the tannic-acid-uranyl-acetate, ("TAURAC") procedure. This new procedure provides remarkably good preservation of native features in relaxed insect flight muscle. We computed 3-D reconstructions from single images of oblique transverse sections. The reconstructions reveal a square profile of the averaged myosin filaments in cross section view, resulting from the symmetrical arrangement of four pairs of myosin heads in each 14.5-nm repeat along the filament. The square profiles form a very regular right-handed helical arrangement along the surface of the myosin filament. Furthermore, TAURAC fixation traps a near complete 38.7 nm labeling of the thin filaments in relaxed muscle marking the left-handed helix of actin targets surrounding the thick filaments. These features observed in an averaged reconstruction encompassing nearly an entire myofibril indicate that the myosin heads, even in relaxed muscle, are in excellent helical register in the A-band.

Animals↗
PubMed1994

Formation of two-dimensional complexes of F-actin and crosslinking proteins on lipid monolayers: demonstration of unipolar alpha-actinin-F-actin crosslinking.

A method is described for forming two-dimensional (2-D) paracrystalline complexes of F-actin and bundling/gelation proteins on positively charged lipid monolayers. These arrays facilitate detailed structural studies of protein interactions with F-actin by eliminating superposition effects present in 3-D bundles. Bundles of F-actin have been produced using the glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase, the cytoskeletal protein erythrocyte adducin as well as smooth muscle alpha-actinin from chicken gizzard. All of the 2-D bundles formed contain F-actin with a 13/6 helical structure. F-actin-aldolase bundles have an interfilament spacing of 12.6 nm and a superlattice arrangement of actin filaments that can be explained by expression of a local twofold axis in the neighborhood of the aldolase. Well ordered F-actin-alpha-actinin 2-D bundles have an interfilament spacing of 36 nm and contain crosslinks 33 nm in length angled approximately 25-35 degrees to the filament axis. Images and optical diffraction patterns of these bundles suggest that they consist of parallel, unipolar arrays of actin filaments. This observation is consistent with an actin crosslinking function at adhesion plaques where actin filaments are bound to the cell membrane with uniform polarity.

Actinin↗
PubMed1994

Dual EBNA1 promoter usage by Epstein-Barr virus in human B-cell lines expressing unique intermediate cellular phenotypes.

The use of different viral promoters for the expression of the EBNA1 gene product appears to be a critical step in the regulation of Epstein-Barr virus latent gene expression and may reflect the extent of differentiation of B-cell hosts. Low-passage Burkitt lymphoma cell lines resemble immature B cells in that they express CD10 (CALLA) and do not express B-cell activation antigens. In these cells, transcription from a promoter located in the BamHI F fragment of the viral genome results in the exclusive expression of EBNA1, referred to as the latency I pattern of viral gene expression. In contrast, high-passage Burkitt lymphoma cells and lymphoblastoid cell lines resemble activated B cells in that they do not express CD10 but do express activation antigens such as CD23. In these cells, the use of two promoters located in the BamHI W and C fragments of the viral genome leads to the expression of all six EBNA gene products (latency III). We have found that four human B-cell lines, DB, LBW2, LBW14, and Josh 7, stably express a pattern of B-cell differentiation antigens intermediate between those found in latency I and latency III cell lines and characterized by the coexpression of CD10 and CD23. The pattern of EBNA1 promoter usage in these cell lines was examined to determine whether their intermediate cellular phenotype was reflected in their patterns of viral gene expression. DB, LBW2, and LBW14 utilize both the BamHI F promoter region and BamHI W promoter region to transcribe the EBNA1 gene. This stable pattern of mixed promoter usage for the expression of the EBNA gene products in B cells has not previously been described. In addition, these three B-cell lines expressed lower levels of the viral latent gene product EBNA2 than those typically observed in latency III cells. The lower levels of activation of viral and cellular promoters known to be regulated by EBNA2 also correlated with the reduced levels of EBNA2 expression in these cells. These included the viral LMP1 and LMP2A promoters and the cellular CD23B promoter. The fourth B-cell line, Josh 7, expressed EBNA1 mRNAs derived from both the BamHI W promoter and BamHI C promoter, similar to latency III cells. The intermediate cellular phenotype in Josh 7 cells appeared to be due, in part, to a deficiency in the expression of viral LMP1.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, CD↗
PubMed1994

Could popping air capsules affect state anxiety?

The present study examined whether popping air capsules affects students' state anxiety. One group of university students were asked to pop air capsules for 5 min. (experimental group); another group did nothing (the control group). Both groups were tested on the State-Trait Anxiety Inventory. Analyses showed that there was a difference in mean State Anxiety scores between the two groups, with the control group having a lower mean than the experimental group, which was contrary to our expectation. The findings are discussed in relation to an earlier published study of the effects of popping air capsules on changing a person's mood.

Adult↗