Plasminogen activator inhibitor type-1-deficient mice have an enhanced IFN-gamma response to lipopolysaccharide and staphylococcal enterotoxin B

Plasminogen activator inhibitor type-1 (PAI-1) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Systemic inflammation is invariably associated with elevated circulating levels of PAI-1, and during human sepsis plasma PAI-1 concentrations predict an unfavorable outcome. Knowledge about the functional role of PAI-1 in a systemic inflammatory response syndrome is highly limited. In this study, we determined the role of endogenous PAI-1 in cytokine release induced by administration of LPS or staphylococcal enterotoxin B (SEB). Both LPS and SEB elicited secretion of PAI-1 into the circulation of normal wild-type (Wt) mice. Relative to Wt mice, PAI-1 gene-deficient (PAI-1(-/-)) mice demonstrated strongly elevated plasma IFN-gamma concentrations after injection of either LPS or SEB. In addition, PAI-1(-/-) splenocytes released more IFN-gamma after incubation with LPS or SEB than Wt splenocytes. Both PAI-1(-/-) CD4+ and CD8+ T cells produced more IFN-gamma upon stimulation with SEB. LPS-induced IFN-gamma release in mice deficient for uPA, the uPA receptor, or tPA was not different from IFN-gamma release in LPS-treated Wt mice. These results identify a novel function of PAI-1 during systemic inflammation, where endogenous PAI-1 serves to inhibit IFN-gamma release by a mechanism that does not depend on its interaction with uPA/uPA receptor or tPA.     

Implications of a 3.472-3.333 Gyr-old subaerial microbial mat from the Barberton greenstone belt, South Africa for the UV environmental conditions on the early Earth

Modelling suggests that the UV radiation environment of the early Earth, with DNA weighted irradiances of about three orders of magnitude greater than those at present, was hostile to life forms at the surface, unless they lived in specific protected habitats. However, we present empirical evidence that challenges this commonly held view. We describe a well-developed microbial mat that formed on the surface of volcanic littoral sediments in an evaporitic environment in a 3.5-3.3Ga-old formation from the Barberton greenstone belt. Using a multiscale, multidisciplinary approach designed to strongly test the biogenicity of potential microbial structures, we show that the mat was constructed under flowing water by 0.25 microm filaments that produced copious quantities of extracellular polymeric substances, representing probably anoxygenic photosynthesizers. Associated with the mat is a small colony of rods-vibroids that probably represent sulphur-reducing bacteria. An embedded suite of evaporite minerals and desiccation cracks in the surface of the mat demonstrates that it was periodically exposed to the air in an evaporitic environment. We conclude that DNA-damaging UV radiation fluxes at the surface of the Earth at this period must either have been low (absorbed by CO2, H2O, a thin organic haze from photo-dissociated CH4, or SO2 from volcanic outgassing; scattered by volcanic, and periodically, meteorite dust, as well as by the upper layers of the microbial mat) and/or that the micro-organisms exhibited efficient gene repair/survival strategies.     

Small bowel image classification using cross-co-occurrence matrices on wavelet domain

This paper presents a novel system to compute the automated classification of wireless capsule endoscope images. Classification is achieved by a classical statistical approach, but novel features are extracted from the wavelet domain and they contain both color and texture information. First, a shift-invariant discrete wavelet transform (SIDWT) is computed to ensure that the multiresolution feature extraction scheme is robust to shifts. The SIDWT expands the signal (in a shift-invariant way) over the basis functions which maximize information. Then cross-co-occurrence matrices of wavelet subbands are calculated and used to extract both texture and color information. Canonical discriminant analysis is utilized to reduce the feature space and then a simple 1D classifier with the leave one out method is used to automatically classify normal and abnormal small bowel images. A classification rate of 94.7% is achieved with a database of 75 images (41 normal and 34 abnormal cases). The high success rate could be attributed to the robust feature set which combines multiresolutional color and texture features, with shift, scale and semi-rotational invariance. This result is very promising and the method could be used in a computer-aided diagnosis system or a content-based image retrieval scheme.     

ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome engineering were evaluated in Arabidopsis plants using Agrobacterium -mediated floral dip transformation. A T-DNA containing the target site for a ZFN pair, that was shown to be active in yeast, was integrated in the Arabidopsis genome. Subsequently, the corresponding pair of ZFN genes was stably integrated in the Arabidopsis genome and ZFN activity was determined by PCR and sequence analysis of the target site. Footprints were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1 and 200 bp and insertions ranging between 1 and 14 bp. We did not observe any toxicity from expression of the ZFNs. In order to obtain ZFN-induced gene-targeting (GT), Arabidopsis plants containing the target site and expressing the ZFN pair were transformed with a T-DNA GT construct. Three GT plants were obtained from ∼3000 transformants. Two of these represent heritable true GT events, as determined by PCR, Southern blot analysis and sequencing of the resulting recombined locus. The third plant showed an ectopic GT event. No GT plants were obtained in a comparable number of transformants that did not contain the ZFNs. Our results demonstrate that ZFNs enhance site-specific mutagenesis and gene-targeting of Agrobacterium T-DNA constructs delivered through floral dip transformation.     

Functional phylogeny relates LET-756 to fibroblast growth factor 9

Fibroblast growth factors (FGFs) are secreted regulatory proteins involved in various developmental processes. In vertebrates, the FGF superfamily comprises 22 members. In non-vertebrates, six FGF genes have been identified in Ciona intestinalis, three in Drosophila melanogaster, and two (let-756 and egl-17) in Caenorhabditis elegans. The core of LET-756 shares a 30-50% sequence identity with the various members of the superfamily. The relationships between vertebrate and non-vertebrate FGFs are not clear. We made chimeric FGFs by replacing the core region of LET-756 by the cores of various mammalian, fly, and worm FGFs. LET-756 deleted in its core region was no longer able to rescue the lethal phenotype of a let-756 null mutant, and only chimeras containing the cores of FGFs 9, 16, and 20 showed rescue capacity. This core contains an internal motif of six amino acid residues (EFISIA) whose deletion or mutation abolished both the rescue activity and FGF secretion in the supernatant of transfected COS-1 cells. Chimera containing the core of C. intestinalis FGF9/16/20, a potential ortholog of FGF9 lacking the complete EFISIA motif, was not able to rescue the lethal phenotype or be secreted. However, the introduction of the EFISIA motif restored both activities. The data show that the EFISIA motif in the core of LET-756 is essential for its biological activity and that FGFs 9, 16, and 20, which contain that motif, are functionally close to LET-756 and may be evolutionary related. This non-classical mode of secretion using an internal motif is conserved throughout evolution.     

Plasma membrane depolarization induced by abscisic acid in Arabidopsis suspension cells involves reduction of proton pumping in addition to anion channel activation, which are both Ca2+ dependent

In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA). Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization. Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization. We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H+ pump inhibitors. Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization. The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases. These two processes are independent because impairing one did not suppress the depolarization. Both processes are however dependent on the [Ca2+]cyt increase induced by ABA since increase in [Ca(2+)](cyt) enhanced anion channels and impaired H(+)-ATPases.     

14-3-3 isoforms and pattern formation during barley microspore embryogenesis

The members of the 14-3-3 isoform family have been shown to be developmentally regulated during animal embryogenesis, where they take part in cell differentiation processes. 14-3-3 isoform-specific expression patterns were studied in plant embryogenic processes, using barley (Hordeum vulgare L.) microspore embryogenesis as a model system. After embryogenesis induction by stress, microspores with enlarged morphology showed higher viability than non-enlarged ones. Following microspore culture, cell division was only observed among the enlarged microspores. Western blot and immunolocalization of three barley 14-3-3 isoforms, 14-3-3A, 14-3-3B and 14-3-3C were carried out using isoform-specific antibodies. The level of 14-3-3C protein was higher in enlarged microspores than in non-enlarged ones. A processed form of 14-3-3A was associated with the death pathway of the non-enlarged microspores. In the early embryogenesis stage, 14-3-3 subcellular localization differed among dividing and non-dividing microspores and the microspore-derived multicellular structures showed a polarized expression pattern of 14-3-3C and a higher 14-3-3A signal in epidermis primordia. In the late embryogenesis stage, 14-3-3C was specifically expressed underneath the L(1) layer of the shoot apical meristem and in the scutellum of embryo-like structures (ELSs). 14-3-3C was also expressed in the scutellum and underneath the L(1) layer of the shoot apical meristem of 21 d after pollination (DAP) zygotic embryos. These results reveal that 14-3-3A processing and 14-3-3C isoform tissue-specific expression are closely related to cell fate and initiation of specific cell type differentiation, providing a new insight into the study of 14-3-3 proteins in plant embryogenesis.     

Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: a genetic predisposition to viral heart disease

Both enteroviral infection of the heart and mutations in the dystrophin gene can cause cardiomyopathy. Little is known, however, about the interaction between genetic and acquired forms of cardiomyopathy. We previously demonstrated that the enteroviral protease 2A cleaves dystrophin; therefore, we hypothesized that dystrophin deficiency would predispose to enterovirus-induced cardiomyopathy. We observed more severe cardiomyopathy, worsening over time, and greater viral replication in dystrophin-deficient mice infected with enterovirus than in infected wild-type mice. This difference appears to be a result of more efficient release of the virus from dystrophin-deficient myocytes. In addition, we found that expression of wild-type dystrophin in cultured cells decreased the cytopathic effect of enteroviral infection and the release of virus from the cell. We also found that expression of a cleavage-resistant mutant dystrophin further inhibited the virally mediated cytopathic effect and viral release. These results indicate that viral infection can influence the severity and penetrance of the cardiomyopathy that occurs in the hearts of dystrophin-deficient individuals.     

Destabilization of pea lectin by substitution of a single amino acid in a surface loop

Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry. Inactivation of ANF is an important element in processing of food. In our study on the stability ofPisum sativum L. lectin (PSL), a conserved hydrophobic amino acid (Val¹⁰³) in a surface loop was replaced with alanine. The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (T _m ) by some 10 °C in comparison with wild-type (wt) PSL, and the denaturation energy (H) is only about 55% of that of wt PSL. Replacement of an adjacent amino acid (Phe¹⁰⁴) with alanine did not result in a significant difference in stability in comparison with wt PSL. Both mutations did not change the sugarbinding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures. The double mutant, PSL V103A/F104A, was produced inEscherichia coli, but could not be isolated in an active (i.e. sugar-binding) form. Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 °C, as judged from haemagglutination assays. These results open the possibility of production of lectins that are activein planta at ambient temperatures, but are inactive and possibly non-toxic at 37 °C in the intestines of mammals.