Binding of transmissible gastroenteritis coronavirus to cell surface sialoglycoproteins

The surface glycoprotein S of transmissible gastroenteritis virus (TGEV) has two binding activities. (i) Binding to porcine aminopeptidase N (pAPN) is essential for the initiation of infection. (ii) Binding to sialic acid residues on glycoproteins is dispensable for the infection of cultured cells but is required for enteropathogenicity. By comparing parental TGEV with mutant viruses deficient in the sialic acid binding activity, we determined the contributions of both binding activities to the attachment of TGEV to cultured cells. In the presence of a functional sialic acid binding activity, the amount of virus bound to two different porcine cell lines was increased sixfold compared to the binding of the mutant viruses. The attachment of parental virus was reduced to levels observed with the mutants when sialic acid containing inhibitors was present or when the cells were pretreated with neuraminidase. In virus overlay binding assays with immobilized cell surface proteins, the mutant virus only recognized pAPN. In addition, the parental virus bound to a high-molecular-mass sialoglycoprotein. The recognition of pAPN was sensitive to reducing conditions and was not dependent on sialic acid residues. On the other hand, binding to the sialic acid residues of the high-molecular-mass glycoprotein was observed regardless of whether the cellular proteins had been separated under reducing or nonreducing conditions. We propose that binding to a surface sialoglycoprotein is required for TGEV as a primary attachment site to initiate infection of intestinal cells. This concept is discussed in the context of other viruses that use two different receptors to infect cells.     

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.     

Pseudomonas aeruginosa displays an epidemic population structure

Bacteria can have population structures ranging from the fully sexual to the highly clonal. Despite numerous studies, the population structure of Pseudomonas aeruginosa is still somewhat contentious. We used a polyphasic approach in order to shed new light on this issue. A data set consisting of three outer membrane (lipo)protein gene sequences (oprI, oprL and oprD), a DNA-based fingerprint (amplified fragment length polymorphism), serotype and pyoverdine type of 73 P. aeruginosa clinical and environmental isolates, collected across the world, was analysed using biological data analysis software. We observed a clear mosaicism in the results, non-congruence between results of different typing methods and a microscale mosaic structure in the oprD gene. Hence, in this network, we also observed some clonal complexes characterized by an almost identical data set. The most recent clones exhibited serotypes O1, 6, 11 and 12. No obvious correlation was observed between these dominant clones and habitat or, with the exception of some recent clones, geographical origin. Our results are consistent with, and even clarify, some seemingly contradictory results in earlier epidemiological studies. Therefore, we suggest an epidemic population structure for P. aeruginosa, comparable with that of Neisseria meningitidis, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise.     

Regulation of alternative oxidase gene expression in soybean

Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 °C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.     

Arteriovenous carboxyhemoglobin difference in critical illness: fiction or fact?

It is still unclear whether the paradoxical arteriovenous carboxyhemoglobin (COHb) difference found in critical illness is due to increased COHb production by the lung, or whether this gradient is caused by technical artifacts using spectrophotometry. In healthy and matched endotoxemic sheep, blood gases were analyzed with a standard ABL 625 and the updated version, an ABL 725. The latter one was accurately calibrated for COHb wavelengths (SAT 100) to eliminate the FCOHb dependency on oxygen tension. All endotoxemic sheep exhibited a hypotensive-hyperdynamic circulation and a pulmonary hypertension. Interestingly, arteriovenous COHb difference occurred in both healthy and endotoxemic sheep (P<0.001 each). Arterial and central venous COHb concentrations determined with the ABL 625 were significantly lower than those measured with the ABL 725 (P<0.001 each). We conclude that (a) arteriovenous COHb difference per se does not reflect critical illness and (b) measurements with an ABL 625 underestimate COHb concentrations.     

Transferrin modifications and lipid peroxidation: implications in diabetes mellitus

Free iron is capable of stimulating the production of free radicals which cause oxidative damage such as lipid peroxidation. One of the most important mechanisms of antioxidant defense is thus the sequestration of iron in a redox-inactive form by transferrin. In diabetes mellitus, increased oxidative stress and lipid peroxidation contribute to chronic complications but it is not known if this is related to abnormalities in transferrin function. In this study we investigated the role of transferrin concentration and glycation. The antioxidant capacity of apotransferrin to inhibit lipid peroxidation by iron-binding decreased in a concentration-dependent manner from 89% at > or = 2 mg/ml to 42% at 0.5 mg/ml. Pre-incubation of apotransferrin with glucose for 14 days resulted in a concentration-dependent increase of glycation: 1, 5 and 13 micromol fructosamine/g transferrin at 0, 5.6 and 33.3 mmol/l glucose respectively, p < 0.001. This was accompanied by a decrease in the iron-binding antioxidant capacity of apotransferrin. In contrast, transferrin glycation by up to 33.3 mmol/l glucose did not affect chemiluminescence-quenching antioxidant capacity, which is iron-independent. Colorimetric evaluation of total iron binding capacity in the presence of an excess of iron (iron/transferrin molar ratio = 2.4) also decreased from 0.726 to 0.696 and 0.585mg/g transferrin after 0, 5.6 and 33.3 mmol/l glucose, respectively, p < 0.01. In conclusion, these results suggest that lower transferrin concentration and its glycation can, by enhancing the pro-oxidant effects of iron, contribute to the increased lipid peroxidation observed in diabetes.     

Dictyostelium cells' cytoplasm as an active viscoplastic body

We applied a recently developed microrheology technique based on colloidal magnetic tweezers to measure local viscoelastic moduli and active forces in cells of Dictyostelium discoideum. The active transport of nonmagnetic beads taken up by phagocytosis was analyzed by single particle tracking, which allowed us to measure the length of straight steps and the corresponding velocities of the movements. The motion consists of a superposition of nearly straight long-range steps (step length in the micrometer range) and local random walks (step widths about 0.1 microm). The velocities for the former type of motion range from 1 to 3 microm/s. They decrease with increasing bead size and are attributed to rapid active transport along microtubuli. The short-range local motions exhibit velocities of less than 0.5 microm/s and reflect the internal dynamics of the cytoplasm. Viscoelastic response curves were measured by application of force pulses with amplitudes varying between 50 pN and 400 pN. Analysis of the response curves in terms of mechanical equivalent circuits yielded cytoplasmic viscosities varying between 10 and 350 Pa s. Simultaneous analysis of the response curves and of the bead trajectories showed that the motion of the beads is determined by the local yield stress within the cytoplasmic scaffold and cisternae, which varies between sigma = 30 Pa and 250 Pa. The motion of intracellular particles is interpreted in terms of viscoplastic behavior and the apparent viscosity is a measure of the reciprocal rate of bond breakage within the cytoplasmatic network. The viscoelastic moduli are interpreted as dynamic quantities which depend sensitively on the amplitude of the forces, and the rate of bond breakage is determined by the Arrhenius-Kramers law with the activation energy being reduced by the work performed by the applied force. In agreement with previous work, we provide evidence that the myosin II-deficient cells exhibit higher yield stresses, suggesting that the function of myosin II as a cross-linker is taken over by the other (non-active) cross-linkers.     

Effects of Sr2+-substitution on the reduction rates of Yz* in PSII membranes--evidence for concerted hydrogen-atom transfer in oxygen evolution

Several groups have recently investigated the kinetic effects of biochemical treatments, site-directed mutagenesis, or substitution of essential cofactors on the stepwise, water-oxidizing chemistry catalyzed by Photosystem II. Consistently, these studies show evidence for a slowing of the final, oxygen-releasing step, S(3) --> S(0), of the catalytic cycle. To a degree, some of this work also shows a slowing of the earlier S-state transitions. To study these processes in more detail, we have investigated the effect of replacing Ca(2+) with Sr(2+)on the rates of the S-state transitions by using time-resolved electron paramagnetic resonance. The results show a slowdown of the last transition in the cycle, consistent with a report from Boussac et al. [Boussac, A., Sétif, P., and Rutherford, A. W. (1992) Biochemistry 31, 1224-1234], and of the earlier S-state transitions as well, which suggests that a common molecular mechanism is at work and that Sr(2+) is less effective than Ca(2+) in supporting it. While the oxidation of Y(z) by P(680)(+) has been extensively studied and can be understood within the context of nonadiabatic electron tunneling combined with rapid, non-rate-limiting proton transfer in the holo-system [Tommos, C., and Babcock, G. T. (2000) Biochim. Biophys. Acta 1458, 199], the reduction of Y(z*) by the Mn cluster cannot be described effectively by a nonadiabatic electron-transfer formalism. This indicates that this reaction is rate limited by processes other than electron tunneling. We discuss our results for Y(z*) reduction and those of others for the activation parameters (E(a), A, KIE, and rates) associated with this process, in terms of both sequential and concerted proton-coupled, electron transfer. Our analysis indicates that concerted hydrogen-atom transfer processes best explain the observed characteristics of the S-state advances.