A bacterial linear motor: cellular and molecular organization of the contractile cytoskeleton of the helical bacterium Spiroplasma melliferum BC3.

The Mollicutes (Mycoplasma, Acholeplasma, and Spiroplasma) are the smallest, simplest and most primitive free-living and self-replicating known cells. These bacteria have evolved from Clostridia by regressive evolution and genome reduction to the range of 5.8 x 10(5)-2.2 x 10(6) basepairs (bp). Structurally, the Mollicutes completely lack cell walls and are enveloped by only a cholesterol containing cell membrane. The Mollicutes contain what can be defined as a bacterial cytoskeleton. The Spiroplasmas are unique in having a well-defined, dynamic, helical cell geometry and a flat, monolayered, membrane-bound cytoskeleton, which follows, intracellularly, the shortest helical line on the cellular coil. By applying cryo-electron-microscopy to whole cells, isolated cytoskeletons and cytoskeletal fibrils and subunits, as well as by selective extraction of cellular components, we determined, at a resolution of approximately 25 A, the cellular and molecular organization of the cytoskeleton. The cytoskeleton is assembled from a 59 kDa protein. The 59 kDa protein, has an equivalent sphere diameter of approximately 50 A. Given the approximately 100 A axial and lateral spacings in the cytoskeletal ribbons and the near-circular shape of the subunit, we suggest that the subunit is a tetramer of 59 kDa monomers; the tetramers assemble further into flat fibrils, seven of which form a flat, monolayered, well-ordered ribbon. The cytoskeleton may function as a linear motor by differential and coordinated length-changes of the fibrils driven by conformational changes of the tetrameric subunits, the shape of which changes from near circular to elliptical. The cytoskeleton controls both the dynamic helical shape and the consequent motility of the cell. A stable cluster of proteins co-purifies with the cytoskeleton. These apparent membrane and membrane-associated proteins may function as anchor proteins.

The structure of mast cell secretory granules in the blind mole rat (Spalax ehrenbergi).

Eosinophils, basophils, and mast cells produce and secrete active substances whose role is to attack invading parasites and protect the host. In this study we use morphometric methods to study mast cells in the blind mole rat (Spalax ehrenbergi). The subterranean and solitary way of life of this species has led to the evolutionary development of special anatomical, morphological, behavioral, and physiological adaptations. Because of its particular lifestyle, the mole rat is less exposed to parasites than other rodents. This could provide a unique model for research into the pathobiology of mast cells. The paracrystalline structure of the mast cell granule content is composed of parallel plates. Diffraction analysis of electron micrographs of thin sections of araldite-embedded tissues indicated that each crystal line plate is a periodic array of parallelograms. The crystal unit cell volume is approximately 930 nm(3), suggesting that each unit cell is composed of one heparin molecule and one to three additional adsorbed proteins. Morphometric data show that characteristics of the secretory granules of mast cells of the blind mole rat resemble those of other rodents. The mast cell unit granule volume in the present study was calculated to be 0.055 microm(3), similar to that of rat peritoneal mast cells.

Stability of body image and body image dissatisfaction in American college students over about the last 15 years.

It is widely assumed that body image dissatisfaction is increasing, particularly in females. We examined data from comparable samples, University of Pennsylvania introductory psychology students, over a span of about 15 years (1983-1984 versus 1995-1998). Ratings of current and ideal body figure were obtained using silhouettes, along with self-reported height and weight. While males always had a much smaller discrepancy between current and ideal than females, levels of dissatisfaction and gender differences in satisfaction have remained the same in these samples. This finding contrasts with the conclusion of a meta-analysis by Feingold and Mazzella in 1998 (Psychological Science 9 (3), 190-195), which indicates an increased difference in body image satisfaction between men and women over the last two decades. Possible accounts for this difference in results are discussed.

The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes.

We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.

Helical perturbations of the flagellar filament: rhizobium lupini H13-3 at 13 A resolution

Flagellar filaments are highly conserved structures in terms of the underlying symmetry of the polymer, subunit domain organization of the flagellin monomer, amino acid composition and primary sequence at the N and C termini. Traditionally, filaments are classified as "plain" or "complex." In complex filaments, the helical lattice is perturbed in a pairwise manner such that the symmetry is reduced along the 6-start helical lines. Both plain (unperturbed) and complex (helically perturbed) components are helically symmetric and share a common lattice. The perturbation in Rhizobium lupini H13-3 results in a subunit composed of a dimer of flagellin. We have generated a approximately 13 A resolution three-dimensional density map of the complex filament of R. lupini H13-3 from low-dose images of negatively stained filaments. Compared to a previous map, which extended to only approximately 25 A resolution and which was generated from only five filaments containing six layer-lines each, the current map is a product of merging 139 data sets containing 66 layer-lines each. The higher resolution and improved signal-to-noise yield a detailed and interpretable density map. The density map is divided into four concentric rings. These amount to two dense cylinders interconnected by low density radial spokes and wrapped by a three-start external winding. The unperturbed component of the map is strikingly similar to the known plain filament maps and, in particular, to that of Caulobacter crescentus. The helically perturbed component contributes mainly to the filaments's exterior (domain D3) where it comprises the tips of the outer domains interconnecting, pairwise, along the 11-start protofilaments and, again, laterally along the 6-start lines forming vertical and horizontal loops. Strong intersubunit connectivity occurs in the D2 shell and in the inner shell which is dominated by 3-start densities. The contribution of the complex component to the radial spokes seems negligible. Copyright 1998 Academic Press.

A fast-freezing device with a retractable environmental chamber, suitable for kinetic cryo-electron microscopy studies.

The design and construction of a fast-freezing device are described. A polycarbonate chamber, in which the humidity and temperature are controlled by microprocessors, slides on a robust chassis guided by ball or Teflon bushings. In its freezing position, the chamber rests on top of the cryogen vessel. The specimen is therefore frozen directly from the experimental conditions within the chamber without exposure to the external environment. After freezing, the chamber, but not the specimen, rises automatically, vacating space for handling the specimen. The chamber, the shutter, and the specimen are all driven pneumatically at an adjustable speed of up to approximately 10 m s-1 and coordinated by either pneumatic or electronic logical gates. Provisions are made for automatic blotting, spraying, and flashing. The chamber is compatible with a liquid nitrogen-cooled copper block assembly for impact (slam) freezing for freeze-substitution and freeze-fracture.

Non-helical perturbations of the flagellar filament: Salmonella typhimurium SJW117 at 9.6 A resolution.

Using a liquid-helium-cooled superconducting electron cryo-microscope, we obtained low-dose images of negatively stained preparations at 4 K and collected structural data to 1/9.6 -1 for flagellar filaments from the strain SJW117 of Salmonella typhimurium (serotype gt). The subunits of this left-handed, straight filament are non-helically perturbed in a pairwise manner. The perturbation corresponds to an alternating conformation in every other row of subunits. These are the 5-start rows and, necessarily, the resulting structure has a seam. The perturbation is not confined to the outside but extends into the structure. We separated the non-symmetric and symmetric parts of the structural data and generated a three-dimensional reconstruction from the latter. The resulting density map is a structure similar in domain organization to the left-handed filament of S. typhimurium SJW1660. Filtered images generated from the non-symmetric component show an ordered and polar structure. The nature of the perturbation was analyzed by model building using a sphere to represent the subunit at low resolution. A lateral shift of approximately 10 degrees mimics the perturbation.

Mollicutes-wall-less bacteria with internal cytoskeletons.

The structure and motility of the Mollicutes (Spiroplasma, Mycoplasma, and Acholeplasma) are briefly reviewed. The data are presented from the perspective of prokaryotic and eukaryotic motors, cytoskeletons, and cell motility. The Mollicutes are eubacteria derived from Clostridia by regressive evolution and genome reduction to produce the smallest and simplest free-living and self-replicating cells. Structurally, the Mollicutes are characterized by a complete lack of a cell wall and the presence of an internal cytoskeleton. Spiroplasma, which are helical cells with a flat, ribbon-like cytoskeleton, are amenable to structural and geometrical analysis. Motility and shape changes can be explained and modeled by the cytoskeleton acting as a linear motor.

Aggressive boys in the classroom: biased attributions or shared perceptions?

This study of 32 aggressive (AG) and 32 nonaggressive (NA) boys applied a social information processing analysis to interactions between children and their teachers. In a cue reading task, AG and NA subjects estimated teacher anger in ambiguous situations where the targets of the teacher behavior were the subjects, NA peers, and AG peers. AG boys predicted that greater teacher anger would be directed toward themselves than did NA boys. However, this could not be interpreted as an attributional bias specific to AG boys, because both NA and AG boys predicted that greater hostility would be directed toward AG boys. Target status was the primary determinant of cue interpretation. AG boys were more likely than NA boys to choose aggressive solutions to problems involving teachers and to judge aggressive solutions to be competent. The results suggested that NA subjects were actually more effective than AG subjects in enacting an aggressive response.

Growing up with Patricia.

Parents of a child with special needs to describe their experiences coping with the various challenges to the family as they grew up together. They discuss coping strategies, shared responsibility, and the trials of dealing with physicians, schools, and social service agencies. They talk about advocating for their child and preparing for the transition to adulthood. Their story is followed by a commentary by a social worker who suggests techniques that pediatricians can use to empower families of children with disabilities to optimize outcome.

Programmable freeze-substitution and cryo-embedding device.

The construction and performance of a modular and fully controllable freeze-substitution device are described. The core of the device consists of a heavy brass block providing a large, stable thermal mass. The block is composed of two perforated plates and a base plate forming nine deep wells, which enable the concomitant substitution of several samples in various substitution fluids, and in large volumes. The wells are surrounded by an isometric network of tunnels through which either liquid nitrogen or hot air can flow. The isometric network enables heat transfer across short uniform distances throughout the entire block's volume, thus minimizing temperature gradients and differences. The temperature of the substitution fluid, rather than that of the metal block, is monitored by a programmable controller, enabling the presetting of any freeze-substitution regime.

Radial mass density functions of vitrified helical specimens determined by scanning transmission electron microscopy: their potential use as substitutes for equatorial data.

Using STEM dark field images, we have determined linear mass densities and radial density profiles of vitrified helical particles. The samples studied are: TMV, RNA-free helical polymers of TMV coat protein (TMV-P), Salmonella typhimurium bacterial flagellar filaments and Escherichia coli pili. The difference between the profiles obtained for TMV and TMV-P shows a maximum at a radius of about 4 nm, corresponding to the RNA in TMV. Of the peaks that are resolved in X-ray diffraction analysis we can resolve the ones for TMV at radii of approximately 4.2 and approximately 6.7 nm and a shoulder at approximately 7.8 nm. Density peaks in bacterial flagellar filaments appear at radii of approximately 4.2, approximately 6.5, approximately 8.5, and approximately 10.5 nm. Accurate mass data can be obtained if the filaments are embedded in ice layers of uniform thickness; their diameters need to be similar to that of the mass standard (TMV) when these data are measured in a comparative manner. Ice layers are often not uniform, and thickness variations are well revealed in STEM dark field. The signal-to-noise ratio and contrast for the transverse projections are lower than those measured for freeze-dried specimens: half an order and one order of magnitude, respectively. The thinnest uniformly thick ice layer still containing a single layer of particles is approximately 10-15 nm thicker than the particles. Radial mass density functions that are directly determined in STEM may have a potential use as substitutes for the unreliable equatorial data in helical reconstructions of TEM bright field images of vitrified specimens.

A three-start helical sheath on the flagellar filament of Caulobacter crescentus.

An unusual feature in preparations of the Caulobacter crescentus flagellar filaments is that some filaments are surrounded by a set of three windings that form a sheath. We provide evidence that the sheath is composed of subunits having a molecular mass of 24,000 Da. We suggest that the sheath could be composed of protofilaments of flagellin wound around the filament.

The rigidity of bacterial flagellar filaments and its relation to filament polymorphism.

We determined and correlated the rigidity of Salmonella typhimurium, Escherichia coli, and Rhizobium lupini flagellar filaments representing various structural and polymorphic states (plain, complex, straight, superhelical, and right- and left-handed). Persistence length, from which the filament's rigidity and other parameters (Young's modulus, bending force constant, buckling persistence length, flexural deformation, and flexural time) were derived, was determined from electron micrographs of isolated, negatively stained filaments. Outer diameters and radii of strong intersubunit connectivity were determined from three-dimensional image reconstructions and radial mass density profiles from scanning transmission electron microscopy. All filaments appear to be highly rigid with no evident correlation with their helical sense or superhelicity. The complex filament of R. lupini is rigid to the extent that it becomes brittle. The overall flexibility of the flagellum seems to stem mainly from the hook and not from the filament. Polymorphism is probably related to the propelling properties and hydrodynamic shape of the filament rather than to its rigidity.

Conformational switching in the flagellar filament of Salmonella typhimurium.

The flagellar filament of the mutant Salmonella typhimurium strain SJW814 is straight, and has a right-handed twist like the filament of SJW1655. Three-dimensional reconstructions from electron micrographs of ice-embedded filaments reveal a flagellin subunit that has the same domain organization as that of SJW1655. Both show slight changes from the domain organization of the subunits from SJW1660, which possesses a straight, left-handed filament. This points to the possible role of changes in subunit conformation in the left-to-right-handed structural transition in filaments. Comparison of the left and right-handed filaments shows that the subunit's orientation and intersubunit bonding appear to change. The orientation of the subunit in the SJW814 filament is intermediate between that of SJW1655 and SJW1660. Its intermediate orientation may explain why the filaments of SJW1655 and SJW1660 are locked in one conformation, whereas the filament of SJW814 can be induced to switch by, for example, changes in pH and ionic strength.

A molecular switch: subunit rotations involved in the right-handed to left-handed transitions of Salmonella typhimurium flagellar filaments.

Using the combined techniques of cryoelectron microscopy and image analysis, we generated three-dimensional reconstructions of flagellar filaments from straight, right-handed (SJW1655-R) and straight, left-handed (SJW1660-L) Salmonella typhimurium mutants, both of which have the same parental strain (SJW1103). In the filaments from SJW1655, all flagellin subunits have the same conformation (R), while in filaments from SJW1660, the subunits are all in the alternate (L) conformation. The difference between the two three-dimensional density maps reveal the structural changes that accompany switching of the flagellin subunits between the two conformations. In going from the R to L state, the subunit undergoes a rotation 30 degrees clockwise about a radial axis and 38 degrees clockwise about a vertical axis, and suffers a 50 degrees bend of the outer, relative to the inner, subunit domain. The intersubunit spacing, along the 11-start protofilaments, changes from 51.6 A in the right-handed filament to 52.1 A in the left-handed filament. In order to produce the correct corkscrew shape in native filaments, the change in contacts that produces this shortening of 0.5 A must occur among the inner domains at a radius of about 30 A. We suggest that the changes in the middle domains of the subunit are the switch that forces changes in the inner domains.

Point mutations that lock Salmonella typhimurium flagellar filaments in the straight right-handed and left-handed forms and their relation to filament superhelicity.

We have determined the nucleotide sequence of two mutant and the parent fliC genes, encoding the protein flagellin (serotype i), of Salmonella typhimurium. The flagellar filaments of the two mutants, SJW1655 and SJW1660, are locked in the straight-right-handed (R) and straight-left-handed (L) conformations, respectively. Their normal, wild-type, parent strain is SJW1103. These mutant strains differ from the wild-type by only one base-pair: the mutation of SJW1655 occurs at nucleotide 1346 in the flagellin gene, changing a C.G pair to T.A (alanine 449 to valine). The mutation of SJW1660 occurs at nucleotide 1277, changing a G.C pair to C.G (glycine 426 to alanine). The resulting amino acid substitutions are near the C terminus predicted to form an alpha-helical coiled coil. The region contains six heptad repeats. Similar alpha-helical segments (three and four repeats long) are present near the N terminus. Alignment of the 17 flagellin sequences available to date confirms the generality of these segments. The mutations are within that portion of the sequence assigned, by proteolytic cleavage, to the middle flagellin domain whose length corresponds to the six heptad repeats found in the sequence (approximately 50 A). We have shown that these mutations are the sole cause of the straight phenotype by replacing the mutated segments with a wild-type one and restoring both superhelicity and motility.

Video-based training of respite care providers: an interactional analysis of presentation format.

We conducted two studies to evaluate a video-based instructional package for training respite care providers and the role of presentation format (viewing the videotapes alone, with a partner, and with structured group training) as a contextual variable. In Study 1, the results of a within-subjects Latin square design nested within a multiple baseline showed that performance during simulated (role-played) respite care situations improved in five of the six skill areas for the 12 trainees following presentation of the videotape, with no differences between presentation formats. Correct responding generalized to respite care situations involving a developmentally disabled child, and in most cases, acquired skills were maintained for up to 6 months. In Study 2, we conducted a clinical replication of Study 1 under conditions more closely approximating those in which the training program would be implemented by respite care agencies. Results of the between-groups analysis were consistent with the findings of Study 1.

Three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus. A flagellin lacking the outer domain and its amino acid sequence lacking an internal segment.

We obtained a three-dimensional reconstruction of the flagellar filament of Caulobacter crescentus CB15 from electron micrographs of negatively stained preparations. The C. crescentus filament appears, both in negative stain and in the frozen-hydrated state, significantly smoother and narrower than other filaments. Its helical symmetry, and unit cell size, however, are similar to that of other filaments. Although the molecular weight of the C. crescentus flagellin is about half that of other plain flagellins, there is only one monomer per unit cell as indicated by diffraction studies and by linear mass density measurements with the scanning transmission electron microscope. Alignment of the primary amino acid sequences of Salmonella typhimurium (serotype i) and C. crescentus (29,000 Mr) flagellins shows that whereas there is homology at the amino and carboxyterminal ends of the two sequences, the central segment of the S. typhimurium sequence has no homology to that of C. crescentus. A correlated comparison between the three-dimensional reconstructions of the two filaments and primary amino acid sequences of the two flagellins suggests that: (1) the C. crescentus subunit is missing the outer molecular domain but is, otherwise, similar to that of S. typhimurium; (2) the outer molecular domain in S. typhimurium corresponds, therefore, to a central stretch of the primary amino acid sequence; and (3) the outer molecular domain, missing in C. crescentus, is not obligatory for flagellar motility.