X-ray diffraction and electron microscopy studies of frozen erythrocyte membrane preparations.

Well-defined X-ray diffraction patterns have been recorded from erythrocyte membranes in the frozen state. At -40 degrees C, lamellar periodicities range from 19 to 95 nm depending on the glycerol content (0--40%, respectively). Freeze-fracture electon micrographs of samples frozen in two stages to approximate to the diffraction conditions show ice formation external to membrane stacks. The membrane stacks have periodicities of the same order of magnitude as those obtained by X-ray diffraction.

Ultrastructure of native lipoprotein from Escherichia coli envelopes.

The free form of the major lipoprotein from Escherichia coli cells envelopes has been purified to homogeneity by gentle extraction procedures and conventional chromatographic separations in a non-ionic detergent. The morphology of paracrystals obtained from homogeneous protein was investigated by low-dose electron microscopy. Electron diffraction of the paracrystals was consistent with alpha-helices arranged perpendicularly to the main cross-band with a periodicity of 20 nm.

Structural studies of cytochrome reductase. Improved membrane crystals of the enzyme complex and crystallization of a subcomplex.

Membrane crystals of mitochondrial ubiquinol: cytochrome c reductase of improved size and long-range order and of the cytochrome bc1 subcomplex have been obtained by a dialysis method. The enzyme--Triton X-100 complex was mixed with Triton phospholipid micelles and the Triton slowly removed by dialysis for 48 hours at pH 5.5 at room temperature or above. The effect of varying the pH and temperature on the shape, size and order of the crystals is described.

Supermarket baker's asthma: how accurate is routine health surveillance?

BACKGROUND: Regular health surveillance is commonly recommended for workers exposed to occupational antigens but little is known about how effective it is in identifying cases. AIMS: To report one large company's surveillance and compare its findings with those of a standard cross-sectional survey in the same workforce. METHODS: A supermarket company with 324 in-store bakeries producing bread from raw ingredients conducted a three-stage health surveillance programme in around 3000 bakery employees. The first stage involved the administration of a simple respiratory questionnaire. If chest symptoms were present a second questionnaire focusing on their work relationship was administered. If positive a blood sample was requested for the measurement of specific IgE to flour and fungal alpha-amylase. The results were compared to an independent cross-sectional survey of employees in 20 of the company's stores. RESULTS: Two hundred and ninety nine (92%) of the company's bakeries took part in surveillance. The overall employee response for the first stage was 77%; a quarter of those with respiratory symptoms reported that they were work related. Seventy four (61%) of those with work related chest symptoms had a measurement of specific IgE to either flour or fungal alpha-amylase, of whom 30 (41%) had a positive result. Surveillance estimated that 1% of bakery employees (1% bakers, 2% managers, 0.6% confectioners) had work related symptoms with specific IgE. This compared with 4% (7.5% bakers, 3.3% managers, 0% confectioners) in the cross-sectional survey (n = 166, 93% response). CONCLUSION: Comparison with a standard cross-sectional survey suggests that routine surveillance can underestimate the workplace burden of disease. The reasons may include technical or resource issues and uncertainties over confidentiality or the perceived consequences of participation. More research needs to be done looking into the design and efficacy of surveillance in occupational asthma.

Humidity and aggregate content correction factors for air-coupled ultrasonic evaluation of concrete.

This paper describes the use of non-contact ultrasound for the evaluation of concrete. Micromachined capacitance transducers are used to transmit ultrasonic longitudinal chirp signals through concrete samples using air as the coupling medium, and a pulse compression technique is then employed for measurement of time of flight through the sample. The effect on the ultrasonic wave speed of storing concrete samples, made with the same water/cement ratio, at different humidity levels is investigated. It is shown that there is a correlation between humidity and speed of sound, allowing a correction factor for humidity to be derived. A strong positive linear correlation between aggregate content and speed of sound was then observed; there was no obvious correlation between compressive strength and speed of sound. The results from the non-contact system are compared with that from a contact system, and conclusions drawn concerning coupling of energy into the samples.

Cytochrome c as a high resolution marker of neurons for light and electron microscopy.

Cytochrome c is taken up by lesioned neurons in the central and peripheral nervous system of the house fly. The enzyme reaction product is evenly distributed in cell bodies, axons, dendrites and axon terminals after 3-6 h uptake. No uptake into undamaged neurons, nor any transneuronal uptake could be demonstrated. Pre- and postsynaptic relations of cytochrome c-labeled neurons can be resolved.

Direct-acting mutagenic properties of some hair dyes used in New Zealand.

Mutagenicity or carcinogenicity data are not publicly available on many hair dyes or dye components commonly used within New Zealand. Representative mid- to dark-warm brown hair dyes of 12 brands supplying more than 1% of the New Zealand market were tested for direct-acting mutagenicity using the bacterial 'Ames' test. Despite recent scientific advances in the development of non-mutagenic dyes, 23 of the 40 products tested gave positive results in one or both of the tester strains used. There appeared to be differences between distributors in the proportion of their hair dyes which were mutagenic. In the case of 6 out of 10 of the above dyes which had tested positive, in vitro mutagenicity or toxicity was enhanced in the presence of verapamil, suggesting that risks from hair-dye exposure may change in the case of persons using this or similar drugs. It is recognised that there are uncertainties regarding human risks from dyes which are shown to be mutagenic in in vitro tests. However, from the above results, it seems possible to produce non-mutagenic hair dyes in this color range; and in the interests of public reassurance, it may be prudent to require that such dyes be used.

In vitro and in vivo mutagenicity studies on sporidesmin, the toxin associated with facial eczema in ruminants.

Sporidesmin, a fungal toxin with widespread distribution within New Zealand, is thought to exert toxic effects through oxidative damage. The purified chemical was tested for its ability to cause point mutations in four strains of Salmonella typhimurium (TA98, TA100, TA102 and TA1537), in the presence and absence of exogenous metabolic activation. Although toxic effects were seen at concentrations exceeding 400 mu gl/plate, there were no significant increases in revertant colonies. In strain TA102, these results were not modified by the presence of glutathione. In AA8 Chinese hamster cells, sporidesmin acted as a potent clastogen, causing chromosomal breaks at concentrations as low as 3 ng/ml, where there was very little reduction in cell viability. Effects were primarily at the chromatid level, but some chromosomal events were also seen. Following low doses, the most common events were chromatid deletions and induction of double minute chromosomes. Interchange events occurred at concentrations of 10 ng/ml and above. The most common of these events was an incomplete chromatid interchange, although some examples of complete chromatid and chromosomal interchange were seen. These in vitro experiments were subsequently extended to an in vivo study of sporidesmin-induced lymphocytic micronuclei (MN) in sheep. In a double blind experiment, 5 sheep were treated with a single high dose of sporidesmin. Blood samples were taken from these, and from 5 untreated sheep, at various intervals before and after treatment. Peripheral blood lymphocytes cultures were harvested and scored for MN in cytokinesis-blocked cells, as a measure of clastogenic activity of sporidesmin in vivo. Following decoding, statistical analysis of the data revealed no significant differences between the MN levels in peripheral blood lymphocytes of sporidesmin-treated and untreated sheep. Although the possibility still exists that clastogenic effects could occur in other species, the data indicate that sporidesmin is not a clastogen in sheep, even though this species is highly susceptible to the toxic effects of sporidesmin.

Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets.

A simple method to determine transient conformations of biological molecules is described. The two reactants (e.g. protein complex and ligand) are mixed rapidly by the coalescence of spray droplets containing one component, with a thin, grid-supported aqueous film containing the other. The transient state is then trapped by rapid freezing, and investigated later by cryo-microscopy. Images of conformations associated with reaction times of 1-100 ms can be achieved by adjusting the delay between the droplet impact and freezing. The droplets (typically 1 micron in diameter) are propelled onto the grid by an atomizer spray. It is shown that the droplets impinging on the liquid film spread rapidly over its surface under the influence of surface tension, and only weakly disturb the underlying film, partially displacing its contents away from the point of impact. Experiments with sprayed salt solutions, using vesicles derived from erythrocytes as micro-osmometers, indicate that rapid mixing occurs both through the film and laterally, by diffusion. The spraying process does not produce any detectable concentration changes due to drying in either the droplets or the film, and the method is applicable to high-resolution imaging.

Methods for specimen thickness determination in electron microscopy. II. Changes in thickness with dose.

The electron diffraction patterns of tilted thin crystals were used to determine the unit cell size in the direction normal to the supporting film. The method revealed a considerable dose-dependent thinning or shrinkage. Using a variety of specimens and stains, we found that this amounted to a 50% reduction in volume and could be attributable to two causes. Firstly, the specimen is held to the supporting film so that volume changes can only occur through changes in thickness. Secondly, the decrease in volume is associated with a dose-induced mass loss which is greatly suppressed at liquid nitrogen temperatures.

Methods for specimen thickness determination in electron microscopy.

A new method is described for specimen thickness determination in transmission electron microscopy. This is carried out by marking specimens with gold particles and analysing the images of a tilt series by computer. The method makes it possible to distinguish populations of particles on different planes and calculate the distance between the planes with statistical variation. We have applied it to carbon films as test objects and compared the results with those obtained by transverse sectioning, STEM mass measurement, optical density and frequency change of a quartz crystal oscillator. We have then used the method for thickness measurement of multilayered protein crystals and thin sectioned cells.

Evaluation of charging on macromolecules in electron cryomicroscopy.

We describe procedures to assess charging of biological specimens under electron irradiation in an electron cryomicroscope. Charging can be observed by an expansion of the illuminating beam, blurring of electron diffraction patterns and by beam "footprints" on the specimen. Discharging can also be seen in the defocused electron diffraction mode. We investigated the influence of a variety of factors on the magnitude and visibility of charging. A reduction of charging is noticed when part of the adjacent carbon film is included in the irradiated specimen area.

Structures of hepatitis B virus cores presenting a model epitope and their complexes with antibodies.

The core shell of hepatitis B virus is a potent immune stimulator, giving a strong neutralizing immune response to foreign epitopes inserted at the immunodominant region, located at the tips of spikes on the exterior of the shell. Here, we analyze structures of core shells with a model epitope inserted at two alternative positions in the immunodominant region. Recombinantly expressed core protein assembles into T=3 and T=4 icosahedral shells, and atomic coordinates are available for the T=4 shell. Since the modified protein assembles predominantly into T=3 shells, a quasi-atomic model of the native T=3 shell was made. The spikes in this T=3 structure resemble those in T=4 shells crystallized from expressed protein. However, the spikes in the modified shells exhibit an altered conformation, similar to the DNA containing shells in virions. Both constructs allow full access of antibodies to the foreign epitope, DPAFR from the preS1 region of hepatitis B virus surface antigen. However, one induces a 10-fold weaker immune response when injected into mice. In this construct, the epitope is less constrained by the flanking linker regions and is positioned so that the symmetry of the shell causes pairs of epitopes to come close enough to interfere with one another. In the other construct, the epitope mimics the native epitope conformation and position. The interaction of native core shells with an antibody specific to the immunodominant epitope is compared to the constructs with an antibody against the foreign epitope. Our findings have implications for the design of vaccines based on virus-like particles.

The changing distribution of occupational asthma: a survey of supermarket bakery workers.

In the UK, since the mid 1980s, supermarkets have accounted for an increasing volume of bread production. Occupational asthma among employees who produce bread from raw ingredients in supermarkets has not been previously investigated. A cross-sectional survey was undertaken involving 239 (71%) employees from 20 different supermarket bakeries. The work-related symptoms were investigated by using questionnaires and measuring the radioallergosorbent test serum-specific immunoglobulin (Ig)E to flour and fungal alpha-amylase. A total of 89 employees underwent whole-shift personal measurement of dust exposure. The geometric mean dust exposure for bakers was 1.2 mg x m(-3), which was higher than for other bakery employees. A total of 37 (15%) employees also reported work-related chest symptoms. Serum IgE to flour was present in 24 (11%) employees and to fungal alpha-amylase in nine (4%) employees. The combination of work-related chest symptoms and specific IgE was found in six (9%) bakers, one (4%) manager and two (3%) assistants. One-quarter of all employees, but half of bakers and managers, had previously worked for different, mainly small, bakeries. This population of bakery workers has important levels of sensitisation and work-related respiratory symptoms, despite low levels of dust exposure. Changes in the location and process of bread manufacture have led to a change in the distribution of bakers' asthma in the UK.

Herpes simplex virus L particles contain spherical membrane-enclosed inclusion vesicles.

The fine structure of light (L) particles of herpes simplex virus type 1 was examined by cryo-electron microscopy and compared to that of virions. The L particles appeared to be spherical entities with significant variation in size, on average smaller in diameter than virions (140 nm compared to 180 nm). The technique confirmed that L particles are composed of an outer envelope, i.e. a bilaminar membrane with protruding glycoprotein spikes, and a uniformly granular tegument, but lack any nucleocapsid. In addition it revealed the presence of one or occasionally more spherical objects, termed inclusion vesicles (IVs), embedded in the tegument of a large proportion of L particles but not observed in virions, suggesting that presence of IVs is unique to the L particles. The IVs vary in size and appear to be composed of a bilaminar membrane without surface projections and filled with material of relatively low electron density, suggesting that the composition of IVs is distinct from that of the envelope and tegument of the L particles.

A special courage: dealing with the Paddington rail crash.

Supporting traumatized employees requires special skills and techniques if it is to be effective. Unfortunately, there is little to inform or guide organizations on how this should be achieved. The present controversy over the use of trauma management systems and debriefing has not been helpful in informing organizations on the best way to take care of employees who become traumatized during the course of their work. This paper looks at how Sainsbury's Supermarkets Ltd managed traumatization through the activation of its Violence at Work policy and procedures, and finally presents the results of an evaluation exercise that was undertaken following the Paddington rail crash.

Low pH induces swiveling of the glycoprotein heterodimers in the Semliki Forest virus spike complex.

Time-resolved cryoelectron microscopy reveals the first step in the conformational changes that enable membrane fusion in Semliki Forest virus. The neutral pH structure reveals a central cavity within the spike complex, plate-like extensions forming a layer above the membrane, and the paths of the paired transmembrane domains connecting the trimeric spikes and pentamer-hexamer clustered capsid subunits. Low pH treatment results in centrifugal movement of E2, the receptor-binding subunit, centripetal movement of E1 to narrow the central cavity initiating the formation of an E1 trimer, and the extension of the E1 fusion sequence toward the target membrane.

Intra-articular apatite deposition in mixed connective tissue disease: crystallographic and technetium scanning characteristics.

An acute arthritis in a patient with mixed connective tissue disease (MCTD) was found to be associated with intra-articular deposition of carbonated hydroxyapatite crystals. A technetium hydroxymethylene diphosphonate bone scan showed intense uptake in the delayed phase scan of the affected joints. Synovial fluid analysis demonstrated uptake of the radiopharmaceutical drug directly onto the crystals.