Focus gradient correction applied to tilt series image data used in electron tomography

The resolution in 3D reconstructions from tilt series is limited to the information below the first zero of the contrast transfer function unless the signal is corrected computationally. The restoration is usually based on the assumption of a linear space-invariant system and a linear relationship between object mass density and observed image contrast. The space-invariant model is no longer valid when applied to tilted micrographs because the defocus varies in a direction perpendicular to the tilt axis and with it the shape of the associated point spread function. In this paper, a method is presented for determining the defocus gradient in thin specimens such as sections and 2D crystals, and for restoration of the images subsequently used for 3D reconstruction. The alignment procedure for 3D reconstruction includes area matching and tilt geometry refinement. A map with limited resolution computed from uncorrected micrographs is compared to a volume computed from corrected micrographs with extended resolution.     

A multiparameter screening assay to assess the cytokine-induced expression of endothelial cell adhesion molecules

Compounds which inhibit endothelial cell inflammatory responses are believed to be of therapeutic value. The cell adhesion molecules E-selectin, ICAM-1, and VCAM-1 play important roles in inflammatory reactions by mediating leukocyte-endothelial interactions. To identify compounds which inhibit the expression of these adhesion molecules following cytokine stimulation we developed an assay which measures E-selectin, ICAM-1, and VCAM-1 in the same experiment. For this, we have taken advantage of the technology of time-resolved fluorimetry, which allows detection of several parameters in parallel, employing anti-E-selectin antibody labeled with europium, and anti-ICAM-1 and anti-VCAM-1 labeled with samarium and terbium, respectively. These antibodies were used to detect the respective antigens in human endothelial cells stimulated with TNFalpha or IL-1beta. In cross-competition assays these antibodies were found to bind specifically to TNF- or IL-1-stimulated cells. This assay, in which three parameters are measured in the same experiment, proved to be robust with signal to noise ratios of 25-35 for E-Selectin, 4-8 for ICAM-1, and 3-9 for VCAM-1. The assay proved to be reproducible in high-throughput screening. The experience with this assay demonstrates that multiple parameters can be measured in an enzyme-linked immunosorbent assay-type assay on cells by using time-resolved fluorimetry. The possibility of obtaining several parameters from one experiment is feasible under high-throughput screening conditions and is of interest for other experimental setups in which the simultaneous measurement of several parameters is desired.     

Where did I take that snapshot? Scene-based homing by image matching

In homing tasks, the goal is often not marked by visible objects but must be inferred from the spatial relation to the visual cues in the surrounding scene. The exact computation of the goal direction would require knowledge about the distances to visible landmarks, information, which is not directly available to passive vision systems. However, if prior assumptions about typical distance distributions are used, a snapshot taken at the goal suffices to compute the goal direction from the current view. We show that most existing approaches to scene-based homing implicitly assume an isotropic landmark distribution. As an alternative, we propose a homing scheme that uses parameterized displacement fields. These are obtained from an approximation that incorporates prior knowledge about perspective distortions of the visual environment. A mathematical analysis proves that both approximations do not prevent the schemes from approaching the goal with arbitrary accuracy, but lead to different errors in the computed goal direction. Mobile robot experiments are used to test the theoretical predictions and to demonstrate the practical feasibility of the new approach. Received: 11 December 1997 / Accepted in revised form: 12 June 1998     

Scavenging of superoxide and hydrogen peroxide in blue‐green algae (cyanobacteria)

Blue‐green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant‐type photosynthetic organisms. Within a single prokaryotic cell, they have uniquely accommodated both oxygenic photosynthesis and aerobic respiration, which are known to produce superoxide and hydrogen peroxide as inevitable byproducts. Two types of superoxide dismutase have been characterized in both N₂‐fixing and non‐N₂‐fixing cyanobacteria, namely cytosolic iron‐containing superoxide dismutase and thylakoid‐bound manganese‐containing superoxide dismutase. No qualitative differences between various cell types (vegetative cells, heterocysts) were found. In contrast to chloroplasts, most of the cyanobacterial species show catalatic activity. From two species the corresponding enzymes have been characterized as typical prokaryotic (bifunctional) catalase‐peroxidases with homologies to cytochrome c peroxidases and ascorbate peroxidases. In addition to catalatic activity, some strains exhibit ascorbate peroxidase activity, but to date there are no reports detailing purification and characterization.
Cyanobacteria were found to contain low intracellular ascorbate concentrations (30‐100 µ M ) and 2‐5 m M glutathione. Both monodehydroascorbate and glutathione reductase activities were detected in most species examined, whereas dehydroascorbate reductase activity was absent. The question as to whether a glutathione‐ascorbate cycle exists in cyanobacteria cannot be answered at present.     

Biological Control of Hedge Bindweed (Calystegia sepium) with Stagonospora convolvuli Strain LA39 in Combination with Competition from Red Clover (Trifolium pratense)

In a maize cropping system where a living green cover suppresses many weeds, Calystegia sepium is able to escape control. In this paper we report the potential for biological control of C. sepium by using the bindweed pathogen Stagonospora convolvuli strain LA39 as a mycoherbicide in combination with competition by the green cover plant Trifolium pratense. In a greenhouse experiment, competition from shoots of T. pratense caused a strong reduction of the biomass of C. sepium, and combined competition from shoots and roots had the same effect. In a second, factorial greenhouse experiment, competition by T. pratense again reduced C. sepium biomass. However, S. convolvuli did not influence the number of leaves or the biomass of C. sepium in the greenhouse even though severe necrosis was observed on inoculated bindweed leaves. In contrast, in a 2-year field study, S. convolvuli caused severe disease and a strong reduction of C. sepium ground coverage in maize. Underseeding with T. pratense had no effect on disease severity, but T. pratense reduced ground coverage by C. sepium at one of eight samplings in the first year. In conclusion, S. convolvuli is useful in the field and, as shown in the greenhouse, a competitive green cover might improve biological control of C. sepium.     

Fate and role of peroxisomes during the life cycle of the yeast Saccharomyces cerevisiae: inheritance of peroxisomes during meiosis

 Sporulation in the yeast Saccharomyces cerevisiae is a meiotic developmental process that occurs in MAT a/MATα heterozygotes in response to nutrient deprivation. Here, the fate and role of peroxisomes during sporulation and germination has been examined by a combination of immunoelectron microscopy and the use of pex mutants defective in peroxisomal functions. Using a green fluorescent protein probe targeted to peroxisomes we show that peroxisomes are inherited through meiosis and that they do not increase in number either during sporulation or spore germination. In addition, there is no requirement for peroxisome degradation prior to spore packaging. Unlike the situation in filamentous fungi, peroxisomes do not proliferate during the yeast life cycle. Functional peroxisomes are dispensable for efficient meiotic development on acetate medium since homozygous Δpex6 diploids sporulated well and produced mature spores that were resistant to diethyl ether. Like haploids, diploid cells can proliferate their peroxisomes in response to oleate as sole carbon source in liquid medium, but under these conditions they do not sporulate. On solid oleate medium, homozygous pex5,Δpex6, and pex7 cells were unable to sporulate efficiently, whereas the wild type was. The results presented here are discussed in terms of the transmission of organelles to progeny cells. Accepted: 19 December 1997     

Physiological oxygen tension modulates the chemically induced mitogenic response of cultured rat hepatocytes

Freshly isolated rat hepatocytes were cultured at periportal- (13% O₂) or perivenous-like (4% O₂) oxygen tension and exposed to subtoxic exposure levels of cyproterone acetate (CPA: 10–330 μM), phenobarbital (PB: 0.75-6 mM), and dimethylsulfoxide (DMSO: 0.1–3.3%) from 24–72 h after seeding. Induced alterations in ploidy, in the number of S-phase cells, the degree of binuclearity, and cellular protein content were determined by twin parameter protein/DNA flow cytometry analysis of intact cells and isolated nuclei. CPA and PB increased whereas DMSO decreased dose dependently the total number of S-phase cells. The changes differed within individual ploidy classes and were modulated by the oxygen tension. CPA increased and DMSO decreased the number of S-phase cells preferentially among the diploid hepatocytes at periportal-like oxygen tension. In contrast, PB increased binuclearity and S-phase cells mainly among the tetraploid hepatocytes at perivenous-like oxygen tension. Cellular protein content increased dose dependently after exposure to the hepatomitogens (CPA, PB) and decreased after exposure to DMSO at both oxygen tensions. Comparison with in vitro data proves that chemicals which interact with cells from the progenitor liver compartment (CPA, DMSO) exert their mitogenic activity best in cultures at periportal-like oxygen tension preferentially in diploid hepatocytes, whereas chemicals which affect cells from the functional compartment show a higher activity at perivenous-like oxygen tension. Physiological oxygen tension seems to be an effective modulator of the proliferative response of cultured rat hepatocytes similar to that expected for periportally or perivenously derived hepatocytes. © 1993 Wiley-Liss, Inc.     

Suppression of virus-specific antibody production by CD8+ class I-restricted antiviral cytotoxic T cells in vivo.

The question of whether virus-induced immunosuppression includes the antibody response against the infecting virus itself was evaluated in a model situation. Transgenic mice expressing the T-cell receptor (TCR) specific for peptide 32-42 of lymphocytic choriomeningitis virus (LCMV) glycoprotein 1 presented by Db reacted with a strong transgenic cytotoxic T-lymphocyte (CTL) response starting on day 3 after infection with a high dose (10(6) PFU intravenously [i.v.]) of the WE strain of LCMV (LCMV-WE); LCMV-specific antibody production in the spleen was suppressed in these mice. Low-dose (10(2) PFU i.v.) infection resulted in an antiviral antibody response comparable to that of the transgene-negative littermates. The induction of suppression of LCMV-specific antibody responses was specifically mediated by CD8+ TCR transgenic CTLs, since the LCMV-8.7 variant virus (which is not recognized by transgenic TCR-expressing CTLs because of a point mutation) did not induce suppression. In addition, treatment with CD8 monoclonal antibody in vivo abrogated suppression. Once suppression had been established, it was found to be nonspecific. The abrogation of antibody responses depended on the relative kinetics of the antibody response involved and the kinetics of the anti-LCMV CTL response. Analysis of T- and B-cell subpopulations showed no significant changes, but immunohistochemical analysis of spleens revealed extensive destruction of follicular organization in lymphoid tissue by day 4 in transgenic mice infected with LCMV-WE but not in those infected with the CTL escape mutant LCMV-8.7. Impairment of antigen presentation rather than of T or B cells was also suggested by adoptive transfer experiments, showing that transferred infected macrophages may improve the anti-LCMV antibody response in LCMV-immunosuppressed transgenic recipients; also, T and B cells from suppressed transgenic mice did respond in irradiated and virus-infected nontransgenic mice with antibody formation to LCMV. Such virus-triggered, T-cell-mediated immunopathology causing the suppression of B cells and of protective antibody responses, including those against the infecting virus itself, may permit certain viruses to establish persistent infections.     

Quantitative analysis of ultrasensitive responses

Ultrasensitive responses are common in cellular information transfer because they allow cells to decode extracellular stimuli in an all-or-none manner. Biochemical responses are usually analyzed by fitting the Hill equation, and the estimated Hill coefficient is taken as a measure of sensitivity. However, this approach is not appropriate if the response under consideration significantly deviates from the best-fit Hill equation. In addition, Hill coefficients greater than unity do not necessarily imply ultrasensitive behaviour if basal activation is significant. In order to circumvent these problems we propose a general method for the quantitative analysis of sensitivity, the relative amplification plot, which is based on the response coefficient defined in metabolic control analysis. To quantify sensitivity globally (i.e. over the whole stimulus range) we introduce the integral-based relative amplification coefficient. Our relative amplification approach can easily be extended to monotonically decreasing, bell-shaped or nonsaturated responses.     

A Ras-like GTPase is involved in hyphal growth guidance in the filamentous fungus Ashbya gossypii

Characteristic features of morphogenesis in filamentous fungi are sustained polar growth at tips of hyphae and frequent initiation of novel growth sites (branches) along the extending hyphae. We have begun to study regulation of this process on the molecular level by using the model fungus Ashbya gossypii. We found that the A. gossypii Ras-like GTPase Rsr1p/Bud1p localizes to the tip region and that it is involved in apical polarization of the actin cytoskeleton, a determinant of growth direction. In the absence of RSR1/BUD1, hyphal growth was severely slowed down due to frequent phases of pausing of growth at the hyphal tip. During pausing events a hyphal tip marker, encoded by the polarisome component AgSPA2, disappeared from the tip as was shown by in vivo time-lapse fluorescence microscopy of green fluorescent protein-labeled AgSpa2p. Reoccurrence of AgSpa2p was required for the resumption of hyphal growth. In the Agrsr1/bud1Delta deletion mutant, resumption of growth occurred at the hyphal tip in a frequently uncoordinated manner to the previous axis of polarity. Additionally, hyphal filaments in the mutant developed aberrant branching sites by mislocalizing AgSpa2p thus distorting hyphal morphology. These results define AgRsr1p/Bud1p as a key regulator of hyphal growth guidance.