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R A Milligan

Publications and source records attributed to R A Milligan.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1998

Influence of gender and socio-economic status on dietary patterns and nutrient intakes in 18-year-old Australians.

This study used two-day diet records to examine dietary behaviours in 504 Australian 18 year-olds in relation to gender, socio-economic status (SES) and national dietary guidelines. Fat intake exceeded 30% of energy in about 80% of subjects and was greater than 40% in about one-quarter. Saturated fat provided more than 10% of dietary energy in more than 90% of participants; less than 1% achieved a polyunsaturated to saturated fat ratio of at least one. The major food groups contributing to fat intake were convenience foods (32% in men, 28% in women) and meat (27% in men, 25% in women). Fibre intake was less than 30 g/day in 93% of women and 77% of men. Intakes of calcium, magnesium, potassium, and vitamins C and A, as a ratio of energy consumption, were greater in women than men, while sodium intake was significantly higher in men. Convenience foods were the greatest contributors to sodium intake (27% in men, 22% in women) followed by meat, bread, and soups and sauces. Greater consumption of cereals, fruit, vegetables and low-fat foods in young women of higher SES was reflected in their nutrient profile with higher intake of fibre and vitamin C and lower intake of fat. Men ate more cereals, meat and sugary foods and less fruit, vegetables and low-fat foods. Only 2.5% of men and 4.1% of women conformed with the health promotion message, widely publicised locally, to eat two fruits and five vegetables daily. Not eating breakfast was associated with lower calcium intake in men and women, and lower iron and fibre in take in women. Achieving behavioural changes in young adults must take into account differences in dietary behaviour related to gender and SES.

Adolescent↗
PubMed1997

Kinetic characterization of brush border myosin-I ATPase.

Brush border myosin-I (BBM-I) is a single-headed unconventional myosin found in the microvilli of intestinal epithelial cells. We used stopped-flow kinetic analysis to measure the rate and equilibrium constants for several steps in the BBM-I ATPase cycle. We determined the rates for ATP binding to BBM-I and brush border actomyosin-I (actoBBM-I), the rate of actoBBM-I dissociation by ATP, and the rates for the steps in ADP dissociation from actoBBM-I. The rate and equilibrium constants for several of the steps in the actoBBM-I ATPase are significantly different from those of other members of the myosin superfamily. Most notably, dissociation of the actoBBM-I complex by ATP and release of ADP from actoBBM-I are both very slow. The slow rates of these steps may play a role in lengthening the time spent in force-generating states and in limiting the maximal rate of BBM-I motility. In addition, release of ADP from the actoBBM-I complex occurs in at least two steps. This study provides evidence for a member of the myosin superfamily with markedly divergent kinetic behavior.

Kinetics↗
PubMed1997

Brush border myosin-I structure and ADP-dependent conformational changes revealed by cryoelectron microscopy and image analysis.

Brush border myosin-I (BBM-I) is a single-headed myosin found in the microvilli of intestinal epithelial cells, where it forms lateral bridges connecting the core bundle of actin filaments to the plasma membrane. Extending previous observations (Jontes, J.D., E.M. Wilson-Kubalek, and R.A. Milligan. 1995. Nature [Lond.]. 378:751-753), we have used cryoelectron microscopy and helical image analysis to generate three-dimensional (3D) maps of actin filaments decorated with BBM-I in both the presence and absence of 1 mM MgADP. In the improved 3D maps, we are able to see the entire light chain-binding domain, containing density for all three calmodulin light chains. This has enabled us to model a high resolution structure of BBM-I using the crystal structures of the chicken skeletal muscle myosin catalytic domain and essential light chain. Thus, we are able to directly measure the full magnitude of the ADP-dependent tail swing. The approximately 31 degrees swing corresponds to approximately 63 A at the end of the rigid light chain-binding domain. Comparison of the behavior of BBM-I with skeletal and smooth muscle subfragments-1 suggests that there are substantial differences in the structure and energetics of the biochemical transitions in the actomyosin ATPase cycle.

Actins↗
PubMed1997

Fine tuning a molecular motor: the location of alternative domains in the Drosophila myosin head.

Myosin isoform sequence variation is likely critical for generating differences in contraction velocity and force production exhibited by the various skeletal muscles in an animal. To examine how myosin heavy chain (MHC) isoform diversity could affect physiological function, we studied the locations of structural differences in the motor domains of muscle MHCs from Drosophila melanogaster. Drosophila has only one muscle Mhc gene. Isoform variation is achieved by alternative splicing of a limited number of exons, clearly delineating the domains of MHC that are critical for muscle-specific functions. There are four alternative regions that contribute to the motor domain of Drosophila myosin. We used the X-ray structure of chicken skeletal S1 as a framework to examine the locations of these four regions. One lies near the ATP-binding pocket in a position where amino acid changes might be expected to modulate entry or exit of the nucleotide. Interestingly, the other three are clustered at the distal end of the molecule, surrounding the reactive cysteine SH1 and the pivot point about which the light chain-containing region swings. These observations underscore the importance of this region, distant from the site of ATP entry and the actin binding interface, as a part of the molecule where modulation of function can be achieved.

Amino Acid Sequence↗
PubMed1997

A model for the microtubule-Ncd motor protein complex obtained by cryo-electron microscopy and image analysis.

Kinesin motors convert chemical energy from ATP hydrolysis into unidirectional movement. To understand how kinesin motors bind to and move along microtubules, we fit the atomic structure of the motor domain of Ncd (a kinesin motor involved in meiosis and mitosis) into three-dimensional density maps of Ncd-microtubule complexes calculated by cryo-electron microscopy and image analysis. The model reveals that Ncd shares an extensive interaction surface with the microtubule, and that a portion of the binding site involves loops that contain conserved residues. In the Ncd dimer, the microtubule-bound motor domain makes intimate contact with its partner head, which is dissociated from the microtubule. This head-head interaction may be important in positioning the dissociated head to take a step to the next binding site on the microtubule protofilament.

Adenosine Triphosphatases↗
PubMed1997

Conformational changes due to calcium-induced calmodulin dissociation in brush border myosin I-decorated F-actin revealed by cryoelectron microscopy and image analysis.

Brush border myosin I (BBMI) is a single-headed molecular motor. Its catalytic domain exhibits extensive sequence homology to the catalytic domain of myosin II, while its tail lacks the coiled-coil nature of myosin II. The BBMI tail domain contains at least three IQ motifs and binds calmodulin. Addition of calcium removes one of these calmodulin light chains, with effects on ATPase activity and motility in in vitro assays. Using the techniques of cryoelectron microscopy and helical image analysis we have calculated three-dimensional (3D) maps of BBMI-decorated actin filaments prepared in the presence and absence of calcium. The 3D maps describe a BBMI catalytic domain that is strikingly similar to the catalytic domain of myosin II subfragment 1 (S1), with the exception of a short amino-terminal region of the heavy chain, which is absent from BBMI. The tail domains of BBMI and S1 are highly divergent in structure, continuing on from their respective motor domains with very different geometries. Addition of calcium to BBMI, and the concomitant loss of a calmodulin light chain, results in an extensive reorganization of mass in the tail domain.

Actins↗
PubMed1997

Three-dimensional structure of Brush Border Myosin-I at approximately 20 A resolution by electron microscopy and image analysis.

Brush Border Myosin-I (BBMI) is a single-headed, unconventional myosin found in the microvilli of intestinal epithelial cells where it forms lateral bridges between the core bundle of actin filaments and the plasma membrane of the microvillus. A three-dimensional (3D) reconstruction of BBMI was made from images of negatively stained, two-dimensional (2D) crystals grown on lipid monolayers formed from mixtures of phosphatidylserine and phosphatidylcholine. The resolution of the 3D map extends to approximately 20 A and allows identification of all of the major structural domains of BBMI. The BBMI molecule is composed of three domains: a globular motor domain, a light-chain-binding domain and a lipid-binding domain. In our map, the putative motor domain is connected to an extended density, which we believe to be the light-chain-binding domain. This long, narrow region has three distinct bends, which may delineate the bound calmodulin light chains. Following the last calmodulin there is density which extends for a short distance across the lipid surface and is presumably the carboxy-terminal lipid-binding domain.

Amino Acid Sequence↗