A nitrilase from a metagenomic library acts regioselectively on aliphatic dinitriles

Several novel nitrilases were selected from metagenomic libraries using cinnamonitrile and a mixture of six different nitriles as substrates. The nitrilase gene nit1 was expressed in Escherichia coli and the resulting protein was further examined concerning its biochemical properties. Nit1 turned out to be an aliphatic nitrilase favoring dinitriles over mononitriles. Stereochemical analysis revealed that Nit1 converted the dinitrile 2-methylglutaronitrile regioselectively. Hydrolysis at the ω-nitrile group of a dinitrile, such as catalyzed by Nit1, leads to ω-cyanocarboxylic acids, which are important precursors for chemical and pharmaceutical products. Nit1 metabolized 2-methylglutaronitrile to the corresponding ω-cyanocarboxylic acid 4-cyanopentanoic acid can be used for the production of the fine chemical 1,5-dimethyl-2-piperidone.     

N1L is an ectromelia virus virulence factor and essential for in vivo spread upon respiratory infection

The emergence of zoonotic orthopoxvirus infections and the threat of possible intentional release of pathogenic orthopoxviruses have stimulated renewed interest in understanding orthopoxvirus infections and the resulting diseases. Ectromelia virus (ECTV), the causative agent of mousepox, offers an excellent model system to study an orthopoxvirus infection in its natural host. Here, we investigated the role of the vaccinia virus ortholog N1L in ECTV infection. Respiratory infection of mice with an N1L deletion mutant virus (ECTVΔN1L) demonstrated profound attenuation of the mutant virus, confirming N1 as an orthopoxvirus virulence factor. Upon analysis of virus dissemination in vivo, we observed a striking deficiency of ECTVΔN1L spreading from the lungs to the livers or spleens of infected mice. Investigating the immunological mechanism controlling ECTVΔN1L infection, we found the attenuated phenotype to be unaltered in mice deficient in Toll-like receptor (TLR) or RIG-I-like RNA helicase (RLH) signaling as well as in those missing the type I interferon receptor or lacking B cells. However, in RAG-1(-/-) mice lacking mature B and T cells, ECTVΔN1L regained virulence, as shown by increasing morbidity and virus spread to the liver and spleen. Moreover, T cell depletion experiments revealed that ECTVΔN1L attenuation was reversed only by removing both CD4(+) and CD8(+) T cells, so the presence of either cell subset was still sufficient to control the infection. Thus, the orthopoxvirus virulence factor N1 may allow efficient ECTV infection in mice by interfering with host T cell function.     

The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies

Morphogenesis of human cytomegalovirus (HCMV) is still only partially understood. We have characterized the role of HCMV tegument protein pUL71 in viral replication and morphogenesis. By using a rabbit antibody raised against the C terminus of pUL71, we could detect the protein in infected cells, as well as in virions showing a molecular mass of approximately 48 kDa. The expression of pUL71, detected as early as 48 h postinfection, was not blocked by the antiviral drug foscarnet, indicating an early expression. The role of pUL71 during virus replication was investigated by construction and analysis of a UL71 stop mutant (TBstop71). The mutant could be reconstituted on noncomplementing cells proving that pUL71 is nonessential for virus replication in human fibroblasts. However, the inhibition of pUL71 expression resulted in a severe growth defect, as reflected by an up to 16-fold reduced extracellular virus yield after a high-multiplicity infection and a small-plaque phenotype. Ultrastructural analysis of cells infected with TBstop71 virus revealed an increased number of nonenveloped nucleocapsids in the cytoplasm, many of them at different stages of envelopment, indicating that final envelopment of nucleocapsids in the cytoplasm was affected. In addition, enlarged multivesicular bodies (MVBs) were found in close proximity to the viral assembly compartment, suggesting that pUL71 affects MVBs during virus infection. The observation of numerous TBstop71 virus particles attached to MVB membranes and budding processes into MVBs indicated that these membranes can be used for final envelopment of HCMV.     

Changes in microbial biomass indices after 10 years of farmyard manure and vegetal fertilizer application to a sandy soil under organic management

The main objective of the second Darmstadt trial was to investigate the effects of vegetal fertilizers on soil properties and crop yield in comparison with farmyard manure. The experiment consisted of seven treatments: (i) inorganic fertilizers, (ii) vegetal organic fertilizers, (iii) vegetal organic fertilizers equivalent to biodynamic preparations, (iv) cattle farmyard manure, (v) cattle farmyard manure with addition of biodynamic preparations, (vi) high level of cattle farmyard manure, and (vii) high level of cattle farmyard manure with biodynamic preparations. The soil properties analyzed were pH, soil organic C, N, P, and S, soil microbial biomass C, N, and P, basal respiration and fungal ergosterol. The application of vegetal fertilizers had slightly negative effects on soil organic C, no effects on crop yield (potato, winter rye) and microbial biomass, but positive effects on ergosterol in comparison with farmyard manure. The increase in ergosterol was caused by straw return in the vegetal, but also in the inorganic fertilizer treatments. The biodynamic preparations did not affect the contents of soil organic C and total N. The low effectiveness of vegetal fertiliser in maintaining soil organic C levels is of particular importance for organic cropping systems and should be examined further under different site conditions.     

Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.     

Two new species of Appendiculella (Meliolaceae) from Panama

Two new species of Meliolaceae (black mildews) are described based on specimens recently collected in western Panama. Appendiculella lozanellae on leaves of Lozanella enantiophylla is the first species of Appendiculella known on Cannabaceae. Appendiculella chiriquiensis on leaves of Cupania guatemalensis is the first record of a species of Appendiculella on Sapindaceae. These species differ from known species on their respective host relationships by the presence of larviform appendages attached to the perithecia, as well as by characteristics of hyphae, appresoria and ascospores. Sequence data of 18S and 28S rDNA are published for A. lozanellae, which becomes the third species of Meliolales and the first species of the genus Appendiculella for which molecular data are available.     

NPY receptors in human cancer: a review of current knowledge

Many peptide hormone receptors are over-expressed in human cancer, permitting an in vivo targeting of tumors for diagnostic and therapeutic purposes. NPY receptors are novel and promising candidates in this field. Using in vitro receptor autoradiography, Y1 and Y2 receptors have been found to be expressed in breast carcinomas, adrenal gland and related tumors, renal cell carcinomas, and ovarian cancers in both tumor cells and tumor-associated blood vessels. Pathophysiologically, tumoral NPY receptors may be activated by endogenous NPY released from intratumoral nerve fibers or tumor cells themselves, and mediate NPY effects on tumor cell proliferation and tumoral blood supply. Clinically, tumoral NPY receptors may be targeted with NPY analogs coupled with adequate radionuclides or cytotoxic agents for a scintigraphic tumor imaging and/or tumor therapy.     

Habitat models for a riparian carabid beetle: their validity and applicability in the evaluation of river bank management

In order to assess the management success of river rehabilitation measurements it is necessary to have representative target species and objective statistical methods. In this study we, tested the validity of habitat suitability models for the riparian carabid beetle Bembidion velox in the evaluation of river bank management along the River Elbe, Germany. On the basis of seven independent data sets from different sites and years we have proven the robustness of logistic regression models with respect to their explanatory and predictive power and their applicability in the field. All models had robust explanatory power and described a strong association of B. velox with semi-terrestrial sandy open soil habitats. Transfers of model results for adult beetles to their larvae and vice versa were highly significant with “sand content” and “stem distance” as the main habitat factors for both life stages. To broaden the local explanatory power towards general predictions we performed model cross-validation in space and time. Spatial transfers produced models with excellent discrimination properties, measured by Area Under Curve (AUC) values of Receiver Operating Characteristics (ROC) plots, independent of sampling designs and trapping methodology. However, the applicability of habitat models for B. velox is defined by the validity period, as the availability of suitable habitats for this species is highly temporally variable and dependent on water level. Model transfers between species also demonstrated that the chosen target species is representative for carabids with similar distribution patterns, as the single species model had high predictive power for the occurrence of a multi-species carabid group.