Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k_cat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.     

Cortical progenitor expansion, self-renewal and neurogenesis-a polarized perspective

Neural stem and progenitor cells giving rise to neurons in developing mammalian neocortex fall into two principal classes with regard to location of mitosis-apical and basal, and into three principal classes in terms of cell polarity during mitosis-bipolar, monopolar, and nonpolar. Insight has been gained into how inheritance of polarized, apical and basal, cell constituents is related to symmetric versus asymmetric divisions of these progenitors, and how this inheritance is linked to their expansion, self-renewal, and neurogenesis. Retention and inheritance of the basal process emerge as key for self-renewal, notably for the monopolar progenitors of prospective gyrencephalic neocortex that undergo asymmetric mitoses at basal locations. The resulting expansion of the neocortex during evolution is proposed to be associated with an increased cone-shape of radial units.     

Survival rates in a small hibernator,the edible dormouse: a comparison across Europe

Understanding how local environmental factors lead to temporal variability of vital rates and to plasticity of life history tactics is one of the central questions in population ecology. We used long‐term capture‐recapture data from five populations of a small hibernating rodent, the edible dormouse Glis glis, collected over a large geographical range across Europe, to determine and analyze both seasonal patterns of local survival and their relation to reproductive activity. In all populations studied, survival was lowest in early summer, higher in late summer and highest during hibernation in winter. In reproductive years survival was always lower than in non‐reproductive years, and females had higher survival rates than males. Very high survival rates during winter indicate that edible dormice rarely die from starvation due to insufficient energy reserves during the hibernation period. Increased mortality in early summer was most likely caused by high predation risk and unmet energy demands. Those effects have probably an even stronger impact in reproductive years, in which dormice were more active. Although these patterns could be found in all areas, there were also considerable differences in average survival rates, with resulting differences in mean lifetime reproductive success between populations. Our results suggest that edible dormice have adapted their life history strategies to maximize lifetime reproductive success depending on the area specific frequency of seeding events of trees producing energy‐rich seeds.     

The significance of lipid composition for membrane activity: new concepts and ways of assessing function

In the last decade or so, it has been realised that membranes do not just have a lipid-bilayer structure in which proteins are embedded or with which they associate. Structures are dynamic and contain areas of heterogeneity which are vital for their formation. In this review, we discuss some of the ways in which these dynamic and heterogeneous structures have implications during stress and in relation to certain human diseases. A particular stress is that of temperature which may instigate adaptation in poikilotherms or appropriate defensive responses during fever in mammals. Recent data emphasise the role of membranes in sensing temperature changes and in controlling a regulatory loop with chaperone proteins. This loop seems to need the existence of specific membrane microdomains and also includes association of chaperone (heat stress) proteins with the membrane. The role of microdomains is then discussed further in relation to various human pathologies such as cardiovascular disease, cancer and neurodegenerative diseases. The concept of modifying membrane lipids (lipid therapy) as a means for treating such pathologies is then introduced. Examples are given when such methods have been shown to have benefit. In order to study membrane microheterogeneity in detail and to elucidate possible molecular mechanisms that account for alteration in membrane function, new methods are needed. In the second part of the review, we discuss ultra-sensitive and ultra-resolution imaging techniques. These include atomic force microscopy, single particle tracking, single particle tracing and various modern fluorescence methods. Finally, we deal with computing simulation of membrane systems. Such methods include coarse-grain techniques and Monte Carlo which offer further advances into molecular dynamics. As computational methods advance they will have more application by revealing the very subtle interactions that take place between the lipid and protein components of membranes - and which are so essential to their function.     

GFP-reporter for a high throughput assay to monitor estrogenic compounds

In vitro test systems using yeast cells are a useful tool for the determination of the estrogenic activity of estrogens, phyto- and xeno-estrogens and can be used for monitoring large sample numbers in a routine analysis procedure. Our conventional transactivation assay functions with an expression plasmid expressing estrogen receptor α (ERα) under the control of a copper-inducible CUP1 promoter and a reporter plasmid expressing β-galactosidase under the control of the vitellogenin estrogen response element (ERE). In the novel yeast screen system the lacZ gene in the reporter plasmid was substituted by a gene for green fluorescent protein (GFP). Incubation of yeast with various concentrations of estrogenically active substances led to expression of the reporter gene product GFP in a dose dependent manner. The yeast transactivation assay was further down-scaled to be performed in a microplate scale, which is an important step to facilitate handling of large sample numbers. The sensitivity and reproducibility of the novel test system could be confirmed by analysis of the potencies of various estrogenically active substances. Thus, the newly developed yeast estrogen screen using GFP as a reporter can substitute the assay that has been used for a period of several years.     

Co-localization of CD3 and prion protein in Jurkat lymphocytes after hypothermal stimulation

While long-term effects of temperature treatment in respect of, e.g., gene-expression and cellular function have already been studied in some detail, nothing is known on the physiological responses of lymphocytes during short-term hypothermal shifts. In this report, we characterized the effects of such a stimulation using the human lymphocyte cell line Jurkat E6.1 and present evidence that warming from 4 to 37 degrees C for only 2 min is sufficient to cause co-localization of CD3, prion protein and the lipid-raft ganglioside GM1 paralleling lymphocyte activation as observed by Ca(2+) mobilization and mitogen-activated protein kinase-phosphorylation.     

Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis <Emphasis Type="Italic">NPR1</Emphasis>

Cotton is an economically important crop worldwide that suffers severe losses due to a wide range of fungal/bacterial pathogens and nematodes. Given its susceptibility to various pathogens, it is important to obtain a broad-spectrum resistance in cotton. Resistance to several fungal and bacterial diseases has been obtained by overexpressing the Non-expressor of Pathogenesis-Related genes-1 (NPR1) in various plant species with apparently minimal or no pleiotropic effects. We examined the efficacy of this approach in cotton by constitutive expression of the Arabidopsis (Arabidopsis thaliana) NPR1 gene. The results show that NPR1-expressing lines exhibited significant resistance to Verticillium dahliae isolate TS2, Fusarium oxysporum f. sp. vasinfectum, Rhizoctonia solani, and Alternaria alternata. Interestingly, the transformants also showed significant resistance to reniform nematodes. Analysis of defense-related, biochemical and molecular responses suggest that when challenged with pathogens or certain systemic acquired resistance-inducing chemicals, the transgenic lines respond to a greater degree compared to the wild-type plants. Importantly, the basal activities of the defense-related genes and enzymes in uninduced transformants were no different than those in their non-transgenic counterparts. The results provide additional evidence supporting the role of NPR1 as an important part of the plant defense system and suggest a means to achieve broad-spectrum resistance to pathogens via genetic engineering.     

Yttrium containing head-on complexes of silico- and germanotungstate: Synthesis, structure and solution properties

Two dimeric head-on complexes of yttrium containing silico- and germanotungstate were isolated from the one-pot reaction of Y(NO₃)₃·6H₂O with the lacunary Na₁₀[MW₉O₃₄]·16H₂O (M = Si and Ge) building blocks in an acetate buffer at pH 4.5. Both polyanions were structurally characterized using various solid-state analytics, such as single-crystal X-ray diffraction, single-crystal X-ray analysis shows that both polyanions crystallize in the monoclinic crystal system (S.G. P2₁/c). FT-IR spectroscopy, or thermogravimetric analysis. The stability of the polyanion in aqueous solution was studied by multinuclear NMR spectroscopy (¹⁸³W, ⁸⁹Y, ²⁹Si, ¹³C, and ¹H). As expected, the ¹⁸³W NMR spectra display six peaks in the intensity ratio of 4:4:2:4:4:4 which indicates that both polyanions exist as dimeric entities in aqueous solution.