Three-dimensional reconstruction of the flagellar hook from Caulobacter crescentus

The structure of the bacterial flagellar hook produced by a mutant of Caulobacter crescentus was studied by electron microscopy, optical diffraction, and digital image processing techniques. The helical surface lattice of the hook is defined by a single, right-handed genetic helix having a pitch of about 23 Å, an axial rise per subunit of 4 Å and an azimuthal angle between subunits of 64·5 °. The lattice is also characterized by intersecting families of 5-start, 6-start and long-pitch 11-start helices. These helical parameters are remarkably similar to those determined for the flagellar filaments from several strains of gram-negative bacteria. The technique of three-dimensional image reconstruction (DeRosier & Klug, 1968) was applied to nine of the better preserved specimens and the diffraction data from five of these were correlated and averaged and used to generate an average three-dimensional model of the hook. The pattern of density modulations in the three-dimensional model is suggestive of an elongated, curved shape for the hook subunit (100 Å × 25 Å × 25 Å). The subunits are situated in the lattice of the polyhook such that their long axes are tilted about 45 ° with respect to the hook axis. The subunits appear to make contact with each other along the 6-start helices at a radius of 80 Å and also along the 11-start helices at a radius of 65 Å. Few structural features are revealed at radii between 15 å and 45 Å and, therefore, we are unable to decide to what extent the hook subunits extend into this region. The most striking characteristic of the model is the presence of deep, broad, continuous 6-start helical grooves extending from an inner radius of about 50 Å to the perimeter of the particle at 105 Å radius. Normal hooks usually appear curved in electron micrographs and sometimes so are the mutant hooks; the prominent 6-start grooves appear to allow for bending with minimal distortion of matter in the outer regions of the hook. A round stain-filled channel about 25 Å in diameter runs down the center of the polyhook. Such a channel supports a model for flagellar assembly in which flagellin subunits travel through the interior of the flagellum to the growing distal end of the filament.     

Eosinophils preserve parasitic nematode larvae by regulating local immunity

Eosinophils play important roles in regulation of cellular responses under conditions of homeostasis or infection. Intestinal infection with the parasitic nematode, Trichinella spiralis, induces a pronounced eosinophilia that coincides with establishment of larval stages in skeletal muscle. We have shown previously that in mouse strains in which the eosinophil lineage is ablated, large numbers of T. spiralis larvae are killed by NO, implicating the eosinophil as an immune regulator. In this report, we show that parasite death in eosinophil-ablated mice correlates with reduced recruitment of IL-4(+) T cells and enhanced recruitment of inducible NO synthase (iNOS)-producing neutrophils to infected muscle, as well as increased iNOS in local F4/80(+)CD11b(+)Ly6C(+) macrophages. Actively growing T. spiralis larvae were susceptible to killing by NO in vitro, whereas mature larvae were highly resistant. Growth of larvae was impaired in eosinophil-ablated mice, potentially extending the period of susceptibility to the effects of NO and enhancing parasite clearance. Transfer of eosinophils into eosinophil-ablated ΔdblGATA mice restored larval growth and survival. Regulation of immunity was not dependent upon eosinophil peroxidase or major basic protein 1 and did not correlate with activity of the IDO pathway. Our results suggest that eosinophils support parasite growth and survival by promoting accumulation of Th2 cells and preventing induction of iNOS in macrophages and neutrophils. These findings begin to define the cellular interactions that occur at an extraintestinal site of nematode infection in which the eosinophil functions as a pivotal regulator of immunity.     

Humanized beta-thalassemia mouse model containing the common IVSI-110 splicing mutation

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.     

Differential effects of hypoxia and acidosis on p53 expression,repair of UVC-damaged DNA and viability after UVC in normal and tumor-derived human cells

Hypoxia and low pH are commonly associated with the tumor microenvironment. We have examined the effects of hypoxia alone (HA) and hypoxia coupled to low pH (HApH) on p53 expression, nucleotide excision repair (NER) and cellular sensitivity to UVC in normal human fibroblasts and human tumor cells. p53 expression was measured using Western blotting, NER using host cell reactivation (HCR) of a UV-damaged reporter gene and cell sensitivity using the MTT assay. HApH resulted in a transient increase in p53 expression in normal fibroblasts at 6 h and in tumor cells at 6–18 h. In normal fibroblasts HApH resulted in a transient increase in HCR at early times (12–24 h) and a concomitant decrease in UVC sensitivity. In contrast, for the tumor-derived cells, the increased HCR of the UVC-treated reporter gene was delayed (36–40 h) and UVC sensitivity increased or remained the same after HApH treatment. These results suggest that early upregulation of p53 and increased repair of UV-damaged DNA after HApH treatment is required for increased cell viability after UVC. HA treatment alone also resulted in a transient increase in HCR of the UVC-damaged reporter gene at early times (12–24 h) in normal fibroblasts and a delayed increase (36–40 h) in the tumor-derived cells. However, the enhanced p53 expression was less or even absent for treatment with HA alone, and HA had no significant affect on cell viability after UVC for any of the cell lines. These results indicate a different cellular response following HApH compared to HA alone.     

Site-specific 3D imaging of cells and tissues with a dual beam microscope

Current approaches to 3D imaging at subcellular resolution using confocal microscopy and electron tomography, while powerful, are limited to relatively thin and transparent specimens. Here we report on the use of a new generation of dual beam electron microscopes capable of site-specific imaging of the interior of cellular and tissue specimens at spatial resolutions about an order of magnitude better than those currently achieved with optical microscopy. The principle of imaging is based on using a focused ion beam to create a cut at a designated site in the specimen, followed by viewing the newly generated surface with a scanning electron beam. Iteration of these two steps several times thus results in the generation of a series of surface maps of the specimen at regularly spaced intervals, which can be converted into a three-dimensional map of the specimen. We have explored the potential of this sequential "slice-and-view" strategy for site-specific 3D imaging of frozen yeast cells and tumor tissue, and establish that this approach can identify the locations of intracellular features such as the 100 nm-wide yeast nuclear pore complex. We also show that 200 nm thick sections can be generated in situ by "milling" of resin-embedded specimens using the ion beam, providing a valuable alternative to manual sectioning of cells and tissues using an ultramicrotome. Our results demonstrate that dual beam imaging is a powerful new tool for cellular and subcellular imaging in 3D for both basic biomedical and clinical applications.     

G541R within the 4070A TM protein regulates fusion in murine leukemia viruses

The mutation G541R within the ectodomain of TM was isolated in three independent chimeric enveloped murine leukemia virus (MuLV) viral populations originally impaired in viral passage and in wild-type 4070A. Isolation of G541R in multiple populations suggested it played a critical role in viral envelope function. Using a viral vector system, the observed effects of the G541R mutation within MuLV envelope proteins were pleiotropic and included effects on the regulation of SU-TM interactions and membrane fusion. G541R suppresses enhanced cell-cell fusion events attributable to the absence of the R-peptide yet does not adversely affect virus titers. The ability to suppress cell-cell fusion is dependent on the presence of the C terminus of the amphotropic 4070A SU protein. Within the wild-type 4070A envelope background, the mutation results in a decreased level of Env at the cell surface that is mirrored in the virion. The TM mutation alters recognition of the SU C terminus by a monoclonal antibody, suggestive of an altered conformation. The presence of G541R allowed the virus to achieve a balance between cytopathogenicity and replication and restored productive viral entry.     

Comparison of metallothionein concentrations and tissue distribution of trace metals in crabs (Pachygrapsus marmoratus) from a metal-rich estuary,in and out of the reproductive season

Crabs, Pachygrapsus marmoratus, were sampled in June 1997 and February 1998 from two sites (at the mouth and 25 km upstream) in the metal-rich Gironde estuary, France. Gills and hepatopancreas were analysed for metal (Cd, Cu, Zn) and metallothionein (MT) contents, in order to examine the influence of both biological and environmental factors on the physico-chemical forms of detoxified metal storage in the crabs. The concentrations of MT and both cytosolic and insoluble metals were not greatly different between males and females, and the influence of organ weights was also minimal. Intersite differences were observed, probably resulting from the gradient of salinity in the estuary, which interacts with both the chemical speciation and bioavailability of metals, and the general protein metabolism of the crabs. Seasonal changes were also important, probably in interaction with the moult and reproductive cycles. In February, concentrations of insoluble metals were generally higher than in June, in both organs, suggesting that essential metals, particularly Zn, are stored during winter then remobilised during the breeding season. The natural variability in the concentrations of MT often concealed any relationship with accumulated metal concentrations. Thus MT in crabs cannot be considered as a useful biomarker of metal pollution.     

Ultrastructural changes in murine lymphocytes induced by aflatoxin B1

This investigation sought to determine whether splenic lymphocytes obtained from Balb/C mice exposed to aflatoxin B1 (AFB1) showed any ultrastructural changes which could account for the immunodysfunction attributable to aflatoxins. Lymphocytes obtained from Balb/C mice administered aflatoxin B1 in olive oil daily for three weeks were studied using both transmission and scanning electron microscopy. The lymphocytes demonstrated ultrastructural changes primarily in the mitochondria where marked internal dissociation of the cristae was revealed by transmission electron microscopy. All other cellular organelles were unaffected. No significant alterations in external structure were observed under scanning electron microscopy. The findings of this study indicate that AFB1 administration does not affect the surface topography of lymphocytes, but AFB1, by causing extensive mitochondrial damage, may affect the way in which these cells function. This could be a possible explanation for the immunodysfunction associated with AFB1.Abbreviations AFB1 Aflatoxin B1 - SEM scanning electron microscopy - TEM transmission electron microscopy