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Biomedical subjects

K A Taylor

Publications and source records attributed to K A Taylor.

At least 19 recordsLinked to original sources
PubMed2001

The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca(2+)-dependent actin binding.

The three-dimensional structure of alpha-actinin from rabbit skeletal muscle was determined by cryoelectron microscopy in combination with homology modeling of the separate domain structures based on results previously determined by X-ray crystallography and nuclear magnetic resonance spectroscopy. alpha-Actinin was induced to form two-dimensional arrays on a positively charged lipid monolayer and micrographs were collected from unstained, frozen hydrated specimens at tilt angles from 0 degrees to 60 degrees. Interpretation of the 15 A-resolution three-dimensional structure was done by manually docking homologous models of the three key domains, actin-binding, three-helix motif and the C-terminal calmodulin-like domains. The initial model was refined quantitatively to improve its fit to the experimental reconstruction. The molecular model of alpha-actinin provides the first view of the overall structure of a complete actin cross-linking protein. The structure is characterized by close proximity of the C-terminal, calmodulin-like domain to the linker between the two calponin-homology domains that comprise the actin-binding domain. This location suggests a hypothesis to explain the involvement of the C-terminal domain in Ca(2+)-dependent actin binding of non-muscle isoforms.

Actinin↗
PubMed2001

The role of pyridoxal phosphate in the function of EspB, a protein secreted by enteropathogenic Escherichia coli.

The sequence of EspB, a secreted protein required for virulence of enteropathogenic Escherichia coli (EPEC), reveals a motif common to enzymes that bind pyridoxal phosphate. Pyridoxal phosphate was not found by fluorometry in concentrated supernatants of EPEC cultures that contain EspB. Plasmids containing cloned espB, in which the lysine residue conserved in the motif was substituted with either an arginine or methionine residue, remained capable of complementing an espB deletion mutant to restore EspB function. The results of these studies do not support a role for pyridoxal phosphate in EspB function.

Actins↗
PubMed2001

Workplace tobacco policy: progress on a winding road.

This is a report of a pre- and post-intervention telephone survey to track changes in workplace tobacco policy in Pima County, Arizona, from 1997 to 1999. During this period, an extensive effort was made to assist workplaces to establish and enforce formal tobacco use policies. A random sample of 1134 workplaces, stratified by workforce size, was surveyed in 1997. Complete interviews were conducted with 934 (82.4%) workplaces. All 934 workplaces were contacted for the follow-up survey that was conducted in 1999. Of these, 824 (88.2%) completed follow-up surveys. The analyses presented were conducted on businesses that were included in both the baseline and follow-up surveys, and that reported having at least one employee on-site (n = 813). Tobacco policies and smoke-free policies were more likely to be found in larger businesses and businesses with a predominantly female workforce. There was no clear policy progression from having no policy, to having a policy, to becoming smoke-free. There was a small but significant overall decrease (4.8%) in the proportion of businesses having policies in the last two years. We found that 10.3% (75) of businesses that had policies in 1997 had dropped their policies by 1999, and that 13.4% (73) of worksites that were smoke-free in 1997 retained tobacco policies but were not smoke-free in 1999. However, formalization of policy in writing and the number of enforcement strategies utilized increased. These findings suggest that efforts to encourage businesses to establish workplace tobacco policies must be sustained even after policies have been established.

Arizona↗
PubMed2001

Enhancing effects of anti-CD40 treatment on the immune response of SCID-bovine mice to Trypanosoma congolense infection.

African trypansosomes are tsetse-transmitted parasites of chief importance in causing disease in livestock in regions of sub-Saharan Africa. Previous studies have demonstrated that certain breeds of cattle are relatively resistant to infection with trypanosomes, and others are more susceptible. Because of its extracellular location, the humoral branch of the immune system dominates the response against Trypanosoma congolense. In the following study, we describe the humoral immune response generated against T. congolense in SCID mice reconstituted with a bovine immune system (SCID-bo). SCID-bo mice infected with T. congolense were treated with an agonistic anti-CD40 antibody and monitored for the development of parasitemia and survival. Anti-CD40 antibody administration resulted in enhanced survival compared with mice receiving the isotype control. In addition, we demonstrate that the majority of bovine IgM+ B cells in SCID-bo mice expresses CD5, consistent with a neonatal phenotype. It is interesting that the percentage of bovine CD5+ B cells in the peripheral blood of infected SCID-bo mice was increased following anti-CD40 treatment. Immunohistochemical staining also indicated increased numbers of Ig+ cells in the spleens of anti-CD40-treated mice. Consistent with previous studies demonstrating high IL-10 production during high parasitemia levels in mice and cattle, abundant IL-10 mRNA message was detected in the spleens and peripheral blood of T. congolense-infected SCID-bo mice during periods of high parasitemia. In addition, although detected in plasma when parasites were absent or low in number, bovine antibody was undetectable during high parasitemia. However, Berenil treatment allowed for the detection of VSG-specific IgG 14 days postinfection in T. congolense-infected SCID-bo mice. Overall, the data indicate that survival of trypanosome-infected SCID-bo mice is prolonged when an agonistic antibody against bovine CD40 (ILA156) is administered. Thus, stimulation of B cells and/or other cell types through CD40 afforded SCID-bo mice a slight degree of protection during T. congolense infection.

Animals↗
PubMed2000

Effects of group B streptococci on cord and adult mononuclear cell interleukin-12 and interferon-gamma mRNA accumulation and protein secretion.

Group B streptococci (GBS) are a major cause of early-onset infection in neonates. Neonates, who have defects in neutrophil function that likely contribute to susceptibility to GBS infection, are deficient in the production of the phagocyte activator interferon (IFN)-gamma. GBS-stimulated mRNA accumulation and protein secretion of IFN-gamma and interleukin (IL)-12, a major enhancer of IFN-gamma production, by mixed mononuclear cells (MMCs) from umbilical cord and adult peripheral blood was examined. GBS-exposed cord blood MMCs secreted lower concentrations of both IL-12 and IFN-gamma proteins than did MMCs from adults. IL-12 and IFN-gamma mRNA accumulation was examined by use of comparative reverse transcriptase-polymerase chain reaction. Cord blood MMCs accumulated less mRNA for both IL-12 and IFN-gamma than did adult blood MMC. The deficiency in cord blood cell production of IL-12 may have a role in inadequate IFN-gamma production, which contributes to the unique susceptibility of neonates to GBS infections.

Adult↗
PubMed2000

Isoforms of alpha-actinin from cardiac, smooth, and skeletal muscle form polar arrays of actin filaments.

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by alpha-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using alpha-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4-0.7 microm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that alpha-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by alpha-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of alpha-actinin.

Actin Cytoskeleton↗
PubMed1999

Visualization of head-head interactions in the inhibited state of smooth muscle myosin.

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.

Animals↗
PubMed1999

Structural studies of cytoskeletal protein arrays formed on lipid monolayers.

Lipid monolayers have been widely used for the production of 2-D crystalline arrays of water-soluble proteins for structural analysis. Less well known is the utility of lipid layers for the assembly of multicomponent structures in two dimensions. This report summarizes current efforts and limitations to utilize a monolayer system composed of the quaternary ammonium surfactant didodecyldimethyammonium and dilaurylphosphatidylcholine to assemble 2-D complexes between actin and cytoskeletal proteins.

Actinin↗
PubMed1999

Tomographic 3D reconstruction of quick-frozen, Ca2+-activated contracting insect flight muscle.

Motor actions of myosin were directly visualized by electron tomography of insect flight muscle quick-frozen during contraction. In 3D images, active cross-bridges are usually single myosin heads, bound preferentially to actin target zones sited midway between troponins. Active attached bridges (approximately 30% of all heads) depart markedly in axial and azimuthal angles from Rayment's rigor acto-S1 model, one-third requiring motor domain (MD) tilting on actin, and two-thirds keeping rigor contact with actin while the light chain domain (LCD) tilts axially from approximately 105 degrees to approximately 70 degrees. The results suggest the MD tilts and slews on actin from weak to strong binding, followed by swinging of the LCD through an approximately 35 degrees axial angle, giving an approximately 13 nm interaction distance and an approximately 4-6 nm working stroke.

Actins↗
PubMed1999

Expression of the EspB protein of enteropathogenic Escherichia coli within HeLa cells affects stress fibers and cellular morphology.

The EspB protein of enteropathogenic Escherichia coli (EPEC) is essential for the signaling events that lead to the accumulation of actin beneath intimately attached bacteria, a process that is known as the attaching and effacing effect. EspB is targeted to the host cell cytoplasm by a type III secretion apparatus. To determine the effect of intracellular EspB on the host cell cytoskeleton, we transfected HeLa cells with a plasmid containing the espB gene under the control of an inducible eukaryotic promoter. A HeLa cell clone that expressed espB mRNA and EspB protein after induction was selected for further study. The expression of EspB in these cells caused a dramatic change in cell morphology and a marked reduction in actin stress fibers. Cells expressing EspB were significantly impaired in their ability to support invasion by EPEC and Salmonella typhimurium. However, the expression of EspB within host cells could not compensate for the lack of EspB expression by an espB mutant strain of EPEC to restore attaching and effacing activity. These studies suggest that EspB is a cytoskeletal toxin that is translocated to the host cell cytoplasm, where it causes a redistribution of actin.

Actins↗
PubMed1998

Immune responses of cattle to African trypanosomes: protective or pathogenic?

Trypanosomosis in domestic livestock negatively impacts food production and economic growth in many parts of the world, particularly in sub-Saharan Africa. Current methods of control are inadequate to prevent the enormous annual socio-economic losses resulting from this disease. Hope for a vaccine based on the variant surface glycoprotein coat was abandoned several years ago when the complexity of the parasite's antigenic repertoire was appreciated. As a result, research is now focused on identifying invariant trypanosome components as potential targets for interrupting infection or infection-mediated disease. The identification of immune mechanisms involved in parasite and disease control, or conversely those responses that are associated with a poor clinical outcome, should facilitate the search for vaccine candidates and subsequent vaccine design strategies. To this end, comparative studies on the immune responses of trypanotolerant and trypanosusceptible breeds of cattle can be exploited. These studies have revealed that trypanotolerant and trypanosusceptible breeds of cattle have distinct antibody responses. Trypanosusceptible cattle produce high titres of polyspecific IgM but fail to produce IgG to specific trypanosome antigens. In contrast, although T cell and macrophage/monocyte responses of infected cattle are depressed, significant differences have not been described between tolerant and susceptible breeds of cattle. In this review, isotype-dependent effector mechanisms, such as complement activation, binding to Fc receptors, activation of phagocytic cells, neutralisation of parasite components, clearance of immune complexes and autoimmune responses, are discussed in the context of their potential impact on either susceptibility or tolerance of cattle to trypanosomosis. In addition, the links between specific cytokine patterns, macrophage/monocyte activation and depressed T cell responses that occur during trypanosome infection are presented. The identification of mechanisms that mediate depressed immune responses might suggest novel disease intervention strategies.

Animals↗
PubMed1998

Characterization of the low magnification performance of a Philips CM300-FEG.

The low magnification performance of a Philips CM300-FEG transmission electron microscope was characterized in three different configurations: CM30-FEG Super Twin, CM30-FEG Twin and CM300-FEG Cryo Twin. Micrographs of gold single crystal, polycrystalline gold, graphitized carbon and copper chloropthalocyanine were recorded in the magnification range 2050x-100,000x using film as the recording medium and optical diffraction to assay image resolution. Lattice images of gold single crystals could be recorded on film with a resolution of 2.04 A at 10,500x; copper chloropthalocyanine lattice images could be recorded with a resolution of 6 A at 4500x and 12 A at 2050x. This indicates that the film has a line resolution as low as 2.1 microns or 470 lines/mm. The fact that lattice images from different specimens can be recorded at this line spacing regardless of magnification suggests that film rather than the microscope is the major limitation to image resolution at low magnification. The image resolution delivered by the microscope is also approximately 4x smaller than the capability of densitometers to digitize it. This suggests that there would be some benefit to digitization of micrographs at finer pixel sizes than currently possible. These observations indicate that tomography of radiation sensitive specimens, where the film is the recording medium, can be recorded at the lowest magnification consistent with digitizing capabilities and desired resolution with confidence in the microscope performance. The potential benefit lies in the possibility of obtaining more images for the same accumulated dose at low magnification and the possibility that more unit cells can be imaged both of which can contribute to the potential success of applying tomographic methods to radiation sensitive specimens.

Carbon↗
PubMed1998

The EspB protein of enteropathogenic Escherichia coli is targeted to the cytoplasm of infected HeLa cells.

The EspB protein of enteropathogenic Escherichia coli (EPEC) is exported via a type III secretion apparatus. EspB is critical for signaling the host cell and for the development of the attaching and effacing lesion characteristic of EPEC infection. We used cellular fractionation and confocal laser scanning microscopy to determine the cellular location of EspB during infection of HeLa cells. Both methods indicated that EspB is targeted to the cytoplasm of infected cells. Using mutants, we found that EspB targeting to the host cell cytoplasm requires the type III secretion apparatus and the secreted proteins EspA and EspD, but not intimin. These results provide insights into the function of the type III secretion apparatus of EPEC and the functions of the Esp proteins.

Bacterial Outer Membrane Proteins↗
PubMed1997

Tomographic three-dimensional reconstruction of insect flight muscle partially relaxed by AMPPNP and ethylene glycol.

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5'-[beta'gamma- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension ("glycol-stiff state"). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of "glycol-stiff" sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90 degrees axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45 degrees angled rigor lead bridges. These 90 degrees "target zone" bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These "nontarget zone" bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90 degrees target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only approximately 25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90 degrees target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements.

Actins↗
PubMed1997

The locus of enterocyte effacement pathogenicity island of enteropathogenic Escherichia coli encodes secretion functions and remnants of transposons at its extreme right end.

The locus of enterocyte effacement (LEE) is necessary for enteropathogenic Escherichia coli to cause characteristic attaching and effacing lesions in host cells. To determine whether sequences at the extreme right end of the LEE downstream of the espB gene are required for attaching and effacing, we constructed a mutant with an omega-interposon insertion immediately downstream of espB. This mutant is incapable of attaching and effacing, of secreting EspA and EspB and of inducing tyrosine phosphorylation of host cell proteins. These phenotypes are restored by a plasmid containing the extreme right end of the LEE. The nucleotide sequence of this region reveals a relatively low G+C content, remnants of transposons, and several open reading frames. The predicted products of these open reading frames include a potential chaperone, a potential component of the secretion apparatus, and a hypothetical peptide with proline rich repeats reminiscent of several eukaryotic proteins. These data indicate that the extreme right end of the LEE is required for attaching and effacing and reveal information relevant to the origin and function of the LEE.

Amino Acid Sequence↗
PubMed1997

The use of electron tomography for structural analysis of disordered protein arrays.

A method based on Fourier transforms is described for obtaining a 3-D reconstruction from a paracrystalline object with static disorder. The method is derived from the standard methods used in 3-D reconstruction of 2-D crystals except that all of the Fourier coefficients are used and not just the sampled data from the periodic lattice. Thus, not only is the spatially ordered part of the structure visualized in 3-D, but also the spatially disordered part. Application of the method to 3-D reconstructions Application of the method to 3-D reconstructions of insect flight muscle is described as well as prospects for extension of the method to radiation-sensitive specimens.

Algorithms↗
PubMed1997

Gender-associated diverse mitochondrial DNA molecules of the mussel Mytilus californianus.

The Pacific-rim, dioecious bivalve Mytilus californianus contains two distinct sequence types of mitochondrial (mt) DNA that are gender-limited in their occurrence. One type (F) is found in both females and males, but the second type (M) is strictly limited to males. Although F- and M-type mtDNAs occur in approximately equal proportion in testes, there is a preponderance of M-type in sperm. Segments of the COI and ND5 genes of F-type and M-type mtDNAs differ in nucleotide sequence by 21.1% and 31.6%, and in predicted amino-acid sequence by 7.9% and 27.1%, respectively. These latter observations raise hitherto unconsidered questions regarding the number of different variants of cytochrome c oxidase and NADH dehydrogenase that may occur in Mytilus non-gametic male cells.

Amino Acid Sequence↗