Automated identification of filaments in cryoelectron microscopy images.

Since the foundation for the three-dimensional image reconstruction of helical objects from electron micrographs was laid more than 30 years ago, there have been sustained developments in specimen preparation, data acquisition, image analysis, and interpretation of results. However, the boxing of filaments in large numbers of images--one of the critical steps toward the reconstruction at high resolution--is still constrained by manual processing even though interactive interfaces have been built to aid the tedious and sometimes inaccurate boxing process. This article describes an accurate approach for automated detection of filamentous structures in low-contrast images acquired in defocus pairs using cryoelectron microscopy. The performance of the approach has been evaluated across various magnifications and at a series of defocus values using tobacco mosaic virus (TMV) preserved in vitreous ice as a test specimen. By integrating the proposed approach into our automated data acquisition and reconstruction system, we are now able to generate a three-dimensional map of TMV to approximately 10-A resolution within 24 h of inserting the specimen grid into the microscope.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

The breastfeeding support team for low-income, predominantly-minority women: a pilot intervention study.

This quasiexperimental pilot study explored whether a focused breastfeeding intervention had potential to improve outcomes in low-income breastfeeding women. Twenty breastfeeding women (10 in intervention and 10 in usual care) were matched on type of delivery, previous breastfeeding experience, and race. Women were low-income, young, 65% high school graduates, and 40% minority. For this intervention, the BST, a breastfeeding support team (community health nurse and peer counselor) provided hospital and home visits and telephone support. Outcomes were measured weekly for the first month, and monthly through month five. At all time periods, more women who received the intervention were breastfeeding. Further, they had less nipple discomfort in the first month; significantly less fatigue in month four and at three and five months reported less fatigue, depression, and anxiety.

Automated image acquisition for single-particle reconstruction using p97 as the biological sample.

We have used Leginon, a fully automatic system capable of acquiring cryo-electron micrographs, to collect data of single particles, specifically of the AAA ATPase p97. The images were acquired under low-dose conditions and required no operator intervention other than the initial setup and periodic refilling of the cold-stage dewar. Each image was acquired at two different defocus values. Two-dimensional projection maps of p97 were calculated from these data and compared to results previously obtained using the conventional manual data collection methods to film. The results demonstrate that Leginon performs as well as an experienced microscopist for the acquisition of single-particle data. The general advantages of automation are discussed.

Leginon: an automated system for acquisition of images from vitreous ice specimens.

We have developed a system to automatically acquire cryo-electron micrographs. The system is designed to emulate all of the decisions and actions of a highly trained microscopist in collecting data from a vitreous ice specimen. These include identifying suitable areas of vitreous ice at low magnification, determining the presence and location of specimen on the grid, automatically adjusting imaging parameters (focus, astigmatism) under low-dose conditions, and acquiring images at high magnification to either film or a digital camera. This system is responsible for every aspect of image acquisition and can run unattended, other than requiring periodic refilling of the cryogens, for over 24 h. The system has been tested out on a variety of specimens that represent typical challenges in the field of cryo-electron microscopy. The results show that the overall performance of the system is equivalent to that of an experienced microscopist.

Breastfeeding duration among low income women.

Breastfeeding has been identified as a possible deterrent to the development of osteoporosis and breast cancer in women. In addition, infants who are breastfed exclusively for at least 4 months reportedly have fewer incidence of SIDS, ear infection, diarrhea, and allergies. Further, low income women who breastfeed may be empowered by the experience. Increasing the frequency and duration of breastfeeding is recognized as a national priority, particularly for low income, minority women. Yet, recent national data indicate that in 1997, only 16.5% of low income mothers breastfed for at least 6 months. Short breastfeeding duration in low income women may be due to problems unique to them; thus, consistent and comprehensive breastfeeding support should be provided by midwives, nurses, lactation consultants, and peer counselors who are skilled in culturally sensitive management of lactation within the context of limited financial and social resources. This article focuses on the benefits of breastfeeding, and factors that may influence its duration. It also explores culturally relevant strategies as well as suggested interventions to increase breastfeeding duration among low-income women.

Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle.

Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins.

A major conformational change in p97 AAA ATPase upon ATP binding.

AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis revealed that a major conformational change of p97 during the ATPase cycle occurred upon nucleotide binding and not during hydrolysis as previously hypothesized. Furthermore, our study indicates that six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners.

Stages of change for health-related behaviours in 18 year-old Australians.

Abstract This study investigated the validity of a Stages of Change algorithm with respect to independent measures of physical activity and fitness. dietary intake and alcohol consumption in 18 year-old Ausmlian men (n = 301) and women (n = 282). Stage of Change categories were related to fat and fibre intakes in men and fibre intake in women as well as hit and vegetable intakes in men and women. Physical activity and fitness for men and women also showed significant linear associations with Stage of Change categories. Alcohol consumption was significantly associated with Stage of Change categories for men but not for women although recorded alcohol consumption was very variable for women. However, the algorithm was valid for both men and women when drinking alcohol consistent with national guidelines on safe drinking was used. In summary, with reference to actual health behaviours, the Stages of Change algorithm was valid for young men and women for diet. physical activity and alcohol drinking. Independent behavioural data were not available for smoking behaviours. Using the algorithm, there were significant associations in men between prccontemplation status for diet and drinking and diet and physical activity, in women between diet and smoking and in both men and women between drinking and smoking. Covariance between precontemplation status for different health behaviours therefore suggests the need for multimodal interventions.

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.

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.

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.

Situs: A package for docking crystal structures into low-resolution maps from electron microscopy.

Three-dimensional image reconstructions of large-scale protein aggregates are routinely determined by electron microscopy (EM). We combine low-resolution EM data with high-resolution structures of proteins determined by x-ray crystallography. A set of visualization and analysis procedures, termed the Situs package, has been developed to provide an efficient and robust method for the localization of protein subunits in low-resolution data. Topology-representing neural networks are employed to vector-quantize and to correlate features within the structural data sets. Microtubules decorated with kinesin-related ncd motors are used as model aggregates to demonstrate the utility of this package of routines. The precision of the docking has allowed for the extraction of unique conformations of the macromolecules and is limited only by the reliability of the underlying structural data.

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.

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.

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.

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.

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.

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.

What happens when fatigue lingers for 18 months after delivery?

OBJECTIVE: To understand the consequences when mothers experience fatigue throughout the first 18 months after birth. DESIGN: Secondary analysis of data from a longitudinal study. Fatigue was measured five times between birth and 18 months after delivery. SETTING: Data for the longitudinal study were collected in different settings (hospital, telephone, and homes). PARTICIPANTS: White mothers who delivered full-term neonates of normal birth weight in a community hospital (N=229). MAIN OUTCOME MEASURES: Persistent fatigue was operationally defined as the report of at least one symptom of fatigue at all five time periods. The association between persistent fatigue and performance outcomes (maternal health, infant health, and infant development) was tested. RESULTS: Results were significant using alpha of .05. Persistent fatigue is associated with perceived maternal health and infant development at 18 months but not infant health. CONCLUSIONS: Findings suggest that persistent fatigue may have a negative effect on performance outcomes for mothers and infants. Assessment for fatigue symptoms should be part of each nursing contact and interpreted as a pattern. Helping mothers choose methods of symptom relief and energy conservation can benefit both the mother and the infant.