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Biomedical subjects

R A Milligan

Publications and source records attributed to R A Milligan.

At least 19 recordsLinked to original sources
PubMed2000

The way things move: looking under the hood of molecular motor proteins.

The microtubule-based kinesin motors and actin-based myosin motors generate motions associated with intracellular trafficking, cell division, and muscle contraction. Early studies suggested that these molecular motors work by very different mechanisms. Recently, however, it has become clear that kinesin and myosin share a common core structure and convert energy from adenosine triphosphate into protein motion using a similar conformational change strategy. Many different types of mechanical amplifiers have evolved that operate in conjunction with the conserved core. This modular design has given rise to a remarkable diversity of kinesin and myosin motors whose motile properties are optimized for performing distinct biological functions.

Actins↗
PubMed1999

A structural change in the kinesin motor protein that drives motility.

Kinesin motors power many motile processes by converting ATP energy into unidirectional motion along microtubules. The force-generating and enzymatic properties of conventional kinesin have been extensively studied; however, the structural basis of movement is unknown. Here we have detected and visualized a large conformational change of an approximately 15-amino-acid region (the neck linker) in kinesin using electron paramagnetic resonance, fluorescence resonance energy transfer, pre-steady state kinetics and cryo-electron microscopy. This region becomes immobilized and extended towards the microtubule 'plus' end when kinesin binds microtubules and ATP, and reverts to a more mobile conformation when gamma-phosphate is released after nucleotide hydrolysis. This conformational change explains both the direction of kinesin motion and processive movement by the kinesin dimer.

Adenosine Triphosphatases↗
PubMed1999

Myosin VI is an actin-based motor that moves backwards.

Myosins and kinesins are molecular motors that hydrolyse ATP to track along actin filaments and microtubules, respectively. Although the kinesin family includes motors that move towards either the plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end of actin filaments. Crystal structures of myosin II (refs 3-6) have shown that small movements within the myosin motor core are transmitted through the 'converter domain' to a 'lever arm' consisting of a light-chain-binding helix and associated light chains. The lever arm further amplifies the motions of the converter domain into large directed movements. Here we report that myosin VI, an unconventional myosin, moves towards the pointed (-) end of actin. We visualized the myosin VI construct bound to actin using cryo-electron microscopy and image analysis, and found that an ADP-mediated conformational change in the domain distal to the motor, a structure likely to be the effective lever arm, is in the opposite direction to that observed for other myosins. Thus, it appears that myosin VI achieves reverse-direction movement by rotating its lever arm in the opposite direction to conventional myosin lever arm movement.

Actins↗
PubMed1999

Structure and function of a membrane-bound murine MHC class I molecule.

MHC molecules are expressed at the surface of nucleated cells to present peptides to T cells. Structural information on MHC molecules has been gathered by x-ray crystallography techniques by using soluble proteins. Although relationships between MHC molecules and cell membranes have not been studied in detail, they are of critical importance for T cell recognition. Using a chemically modified lipid, we have been able to capture and orient histidine-tagged MHC molecules on lipid membranes. Surface plasmon resonance experiments show that the protein binds to the nickel lipid in a specific manner and in an oriented fashion, which allows T cell receptor binding. Similar lipid surfaces have been used to grow two-dimensional crystals and to determine the structure of a membrane-anchored murine H-2Kb MHC class I molecule. The docking of the crystallographic structure into the three-dimensional reconstructed structure derived from the two-dimensional crystals allows us to determine that the histidine tag is near the membrane surface and that the MHC molecule is in an upright position, exposing the peptide/alpha1-alpha2 domains toward the T cell.

Animals↗
PubMed1999

The motor protein myosin-I produces its working stroke in two steps.

Many types of cellular motility, including muscle contraction, are driven by the cyclical interaction of the motor protein myosin with actin filaments, coupled to the breakdown of ATP. It is thought that myosin binds to actin and then produces force and movement as it 'tilts' or 'rocks' into one or more subsequent, stable conformations. Here we use an optical-tweezers transducer to measure the mechanical transitions made by a single myosin head while it is attached to actin. We find that two members of the myosin-I family, rat liver myosin-I of relative molecular mass 130,000 (M(r) 130K) and chick intestinal brush-border myosin-I, produce movement in two distinct steps. The initial movement (of roughly 6 nanometres) is produced within 10 milliseconds of actomyosin binding, and the second step (of roughly 5.5 nanometres) occurs after a variable time delay. The duration of the period following the second step is also variable and depends on the concentration of ATP. At the highest time resolution possible (about 1 millisecond), we cannot detect this second step when studying the single-headed subfragment-1 of fast skeletal muscle myosin II. The slower kinetics of myosin-I have allowed us to observe the separate mechanical states that contribute to its working stroke.

Actins↗
PubMed1999

Motor protein decoration of microtubules grown in high salt conditions reveals the presence of mixed lattices.

We have used back-projection methods to obtain three-dimensional maps of motor-protein decorated nine and ten protofilament microtubules polymerized in the presence of high salt and preserved in vitreous ice. The resulting three-dimensional maps show that the vast majority of these microtubules have multiple seams, rather than being helical as would be expected according to the lattice accommodation model. These results indicate that microtubules should be analyzed by back-projection before using helical reconstruction approaches, and that nine and ten protofilament microtubules polymerized in high salt conditions are not suitable for helical analysis.

Cryopreservation↗
PubMed1999

Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.

gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

Amino Acid Sequence↗
PubMed1999

High-resolution model of the microtubule.

A high-resolution model of the microtubule has been obtained by docking the crystal structure of tubulin into a 20 A map of the microtubule. The excellent fit indicates the similarity of the tubulin conformation in both polymers and defines the orientation of the tubulin structure within the microtubule. Long C-terminal helices form the crest on the outside of the protofilament, while long loops define the microtubule lumen. The exchangeable nucleotide in beta-tubulin is exposed at the plus end of the microtubule, while the proposed catalytic residue in alpha-tubulin is exposed at the minus end. Extensive longitudinal interfaces between monomers have polar and hydrophobic components. At the lateral contacts, a nucleotide-sensitive helix interacts with a loop that contributes to the binding site of taxol in beta-tubulin.

Crystallography, X-Ray↗
PubMed1999

Health promotion in couples adapting to a shared lifestyle.

In a pilot health promotion program for couples, we aimed to build on re-evaluation of attitudes to health occurring early in marriage, and social support provided by partners, to address the weight gain and physical inactivity which may follow marriage. A randomized controlled trial lasting 16 weeks used six modules focusing on nutrition and physical activity but including information about alcohol and smoking. Thirty-four of 39 couples enrolled completed the study. Self-efficacy for diet and physical activity increased significantly in the program group while ranking of barriers to healthy behaviours decreased and ranking of beliefs about the benefits of health behaviours increased relative to controls. Intake of fat, take-away foods and alcohol decreased, and consumption of fruit, vegetables and reduced-fat foods increased significantly in the program group. Physical activity in the program group increased by the equivalent of 50 min of brisk walking weekly but did not differ significantly from controls. Cholesterol fell significantly by 6% more in the program group than controls. In focus groups, participants unanimously found the program valuable. Health promotion programs designed for couples can achieve short-term changes in behaviour and risk factors. Larger trials with longer-term monitoring, incorporating feedback from focus groups and cost-benefit analysis, are in progress.

Adult↗
PubMed1999

Developing a shared language: interdisciplinary communication among diverse health care professionals.

Multidisciplinary teams of nurses, physicians, and other professionals may have difficulty communicating because of inconsistent theoretical underpinnings. A theoretical base that spans both clinical outcomes and professional boundaries is needed. The web of causation is a theoretical framework that provides a platform of communication connecting issues related to infant mortality among various health-related professions. It includes professional, community, and institutional issues relevant to pregnant women and new mothers as infant caregivers. The article discusses how the web was used for interdisciplinary health care professional interaction and how it was used to develop a series of research protocols that will affect the care of mothers and infants in the District of Columbia.

Clinical Protocols↗
PubMed1998

Self-organizing neural networks bridge the biomolecular resolution gap.

Topology-representing neural networks are employed to generate pseudo-atomic structures of large-scale protein assemblies by combining high-resolution data with volumetric data at lower resolution. As an application example, actin monomers and structural subdomains are located in a three-dimensional (3D) image reconstruction from electron micrographs. To test the reliability of the method, the resolution of the atomic model of an actin polymer is lowered to a level typically encountered in electron microscopic reconstructions. The atomic model is restored with a precision nine times the nominal resolution of the corresponding low-resolution density. The presented self-organizing computing method may be used as an information-processing tool for the synthesis of structural data from a variety of biophysical sources.

Actins↗
PubMed1998

Lipid nanotubes as substrates for helical crystallization of macromolecules.

A general approach for crystallization of proteins in a fast and simple manner would be of immense interest to biologists studying protein structure-function relationships. Here, we describe a method that we have developed for promoting the formation of helical arrays of proteins and macromolecular assemblies. Electron micrographs of the arrays are suitable for helical image analysis and three-dimensional reconstruction. We show that hydrated mixtures of the glycolipid galactosylceramide (GalCer) and derivatized lipids or charged lipids form unilamellar nanotubules. The tubules bind proteins in a specific manner via high affinity ligands on the polar head groups of the lipid or via electrostatic interactions. By doping the GalCer with a novel nickel-containing lipid, we have been able to form helical arrays of two histidine-tagged proteins. Similarly, doping with a biotinylated lipid allows crystallization of streptavidin. Finally, three proteins with affinity for positively or negatively charged lipid layers formed helical arrays on appropriately charged tubules. The generality of this method may allow a wide variety of proteins to be crystallized on lipid nanotubes under physiological conditions.

Animals↗
PubMed1998

Three-dimensional structure of Acanthamoeba castellanii myosin-IB (MIB) determined by cryoelectron microscopy of decorated actin filaments.

The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a "classical" myosin-I, Acanthamoeba myosin-IB (MIB), at approximately 18 A resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, approximately 10 degrees, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.

Acanthamoeba↗
PubMed1998

Parental smoking and risk factors for cardiovascular disease in 10- to 12-year-old children.

OBJECTIVE: Smokers have multiple adverse health-related behaviors and an increased risk of cardiovascular disease. We examined whether health behaviors in parents who smoke may influence children's health behaviors. STUDY DESIGN: Cross-sectional data from 10- to 12-year-olds (n = 800) entering a trial of health promotion programs. RESULTS: Smoking in children was independently associated with maternal (odds ratio 2.1, confidence interval 1.2, 3.8) and paternal smoking (odds ratio 2.1, confidence interval 1.2, 3.7) and was less likely in girls (odds ratio 0.4, confidence interval 0.2, 0.6). Maternal smoking and paternal smoking were additive predictors in children of lower physical activity (P = .0013 for mothers; P = .0476 for fathers) and more television watching (P = .0335 for mothers; P = .0241 for fathers). Children's fat intake was significantly greater if either parent smoked. Children's body mass index (P = .0183) and waist-to-hip ratio (P = .0009) were significantly greater if mothers smoked. CONCLUSIONS: Poor health behaviors associated with smoking in parents, particularly mothers, are likely to influence children's long-term risk of having lifestyle diseases. The results may also explain some of the apparent effects attributed to passive smoking in families.

Adult↗
PubMed1998

A controlled trial of health promotion programs in 11-year-olds using physical activity "enrichment" for higher risk children.

OBJECTIVE: To evaluate the short and long term benefits of a school and home based physical activity "enrichment" program for children at higher risk of cardiovascular disease as identified by cluster analysis. STUDY DESIGN: During two 10-week school terms, 800 11-year-olds took part in a randomized controlled trial with the standard physical activity and nutrition program in six schools, the standard program in a further seven schools but with the addition of physical activity enrichment for higher risk children in those schools, and no program in five control schools. Cluster analysis identifying the 29% or so highest risk children used systolic blood pressure, percent body fat, physical fitness, and blood cholesterol. RESULTS: Fitness improved significantly in program schools, particularly with enrichment in higher risk boys. Substantial improvements persisted 6 months later in girls from program schools. At "Enrichment" schools, cholesterol showed significant benefits in higher risk girls and, 6 months later, in both boys and higher risk girls. Sodium intake and, in girls, subscapular skinfolds were lower in "Enrichment" schools when the program ended, but not 6 months later. CONCLUSION: Two-semester health programs with physical activity enrichment for higher risk children can produce benefits sustained for at least 6 months. Improvements extend to lower risk children exposed indirectly to the enrichment. Attenuation of effects on diet and body composition in the longer-term suggest the need for on-going programs.

Anthropometry↗
PubMed1998

First stage labor management: An examination of patterned breathing and fatigue.

BACKGROUND: Patterned breathing is one way that women cope with labor. Fatigue is a frequently reported symptom over which women and caregivers have little control. The purpose of this study was to examine the relationship between the use of patterned breathing, a traditional intervention, and the level of fatigue reported during the first stage of labor. METHOD: A secondary analysis was conducted on a subset (n = 56) of a prospective longitudinal study of fatigue during the intrapartum period. The sample comprised primiparous women in labor whose fatigue was measured every two hours for six hours after admission. At each data point the investigator evaluated the method of breathing that participants used. RESULTS: During the latent phase of labor, women using patterned breathing exhibited significantly more fatigue. In the active phase, differences between groups were not significant. Controlling for age, education, and marital status of participants did not change the results. CONCLUSIONS: It is appropriate for nurses, midwives, physicians, and doulas to encourage the use of patterned breathing as an intervention in active labor; however, patterned breathing may increase the mother's fatigue level if begun too early.

Adaptation, Psychological↗
PubMed1998

Family history as a predictor of blood pressure in a longitudinal study of Australian children.

BACKGROUND: Sex both of parent and of child might influence associations between parental hypertension and blood pressure in offspring. OBJECTIVE: To examine these associations. DESIGN: A cohort of Australians was surveyed 3-yearly from age 9 to 18 years. SETTING: A community-based sample. PARTICIPANTS: When they were aged 18 years, 630 of 1565 participants who had been selected randomly at the age of 9 years were re-surveyed. MAIN OUTCOME MEASURES: Systolic and diastolic blood pressures. RESULTS: Paternal hypertension was reported by 18% of men and 15% of women and maternal hypertension by 15% of men and 14% of women. By the time they were aged 9 years, systolic blood pressure was significantly higher in sons [117.8 mmHg, 95% confidence interval (CI) 116A-119.2 versus 114.7 mmHg, CI 113.4-116.0] and daughters (118.2 mmHg, CI 116.9-119.5 versus 114.9 mmHg, CI 112.8-117.0) of hypertensive fathers than it was in sons and daughters of normotensive fathers. When they were aged 18 years, paternal hypertension predicted blood pressures in men and women independently of their weight at birth, fitness, alcohol consumption and weight for height for age. Systolic blood pressures increased more rapidly (by 0.6 mmHg/year) in men with hypertensive fathers. CONCLUSIONS: Systolic blood pressure in young adults differs in relation to parental hypertension according to the sex of the affected parent and the sex of the offspring. This could reflect unmeasured environmental variables or the action of sex-related genetic or intrauterine factors.

Adolescent↗