Characterization and reconstitution of a 4Fe-4S adenylyl sulfate/phosphoadenylyl sulfate reductase from Bacillus subtilis

CysH1 from Bacillus subtilis encodes a 3'-phospho/adenosine-phosphosulfate-sulfonucleotide reductase (SNR) of 27 kDa. Recombinant B. subtilis SNR is a homodimer, which is bispecific and reduces adenylylsulfate (APS) and 3'-phosphoadenylylsulfate (PAPS) alike with thioredoxin 1 or with glutaredoxin 1 as reductants. The enzyme has a higher affinity for PAPS (K(m)PAPS 6.4 microm Trx-saturating, 10.7 microm Grx-saturating) than for APS (K(m) APS 28.7 microm Trx-saturating, 105 microm Grx-saturating) at a V(max) ranging from 280 to 780 nmol sulfite mg(-1) min(-1). The catalytic efficiency with PAPS as substrate is higher by a factor of 10 (K(cat)/K(m) 2.7 x 10(4)-3.6 x 10(4) liter mol(-1) s(-1). B. subtilis SNR contains one 4Fe-4S cluster per polypeptide chain. SNR activity and color were lost rapidly upon exposure to air or upon dilution. M?ssbauer and absorption spectroscopy revealed that the enzyme contained a 4Fe-4S cluster when isolated, but degradation of the 4Fe-4S cluster produced an inactive intermediate with spectral properties of a 2Fe-2S cluster. Activity and spectral properties of the 4Fe-4S cluster were restored by preincubation of SNR with the iron-sulfur cluster-assembling proteins IscA1 and IscS. Reconstitution of the 4Fe-4S cluster of SNR did not affect the reductive capacity for PAPS or APS. The interconversion of the clusters is thought to serve as oxygen-sensitive switch that suppresses SO(3) formation under aerobiosis.     

Semaphorin 3d promotes cell proliferation and neural crest cell development downstream of TCF in the zebrafish hindbrain

Neural crest cells (NCCs) are pluripotent migratory cells that are crucial to the development of the peripheral nervous system, pigment cells and craniofacial cartilage and bone. NCCs are specified within the dorsal ectoderm and undergo an epithelial to mesenchymal transition (EMT) in order to migrate to target destinations where they differentiate. Here we report a role for a member of the semaphorin family of cell guidance molecules in NCC development. Morpholino-mediated knockdown of Sema3d inhibits the proliferation of hindbrain neuroepithelial cells. In addition, Sema3d knockdown reduces markers of migratory NCCs and disrupts NCC-derived tissues. Similarly, expression of a dominant-repressor form of TCF (DeltaTCF) reduces hindbrain cell proliferation and leads to a disruption of migratory NCC markers. Moreover, expression of DeltaTCF downregulates sema3d RNA expression. Finally, Sema3d overexpression rescues reduced proliferation caused by DeltaTCF expression, suggesting that Sema3d lies downstream of Wnt/TCF signaling in the molecular pathway thought to control cell cycle in NCC precursors.     

Vaspin gene expression in human adipose tissue: association with obesity and type 2 diabetes

Recently, vaspin was identified as an adipokine with insulin-sensitizing effects, which is predominantly secreted from visceral adipose tissue in a rat model of type 2 diabetes. In this study, we examined whether vaspin mRNA expression is a marker of visceral obesity and correlates with anthropometric and metabolic parameters in paired samples of visceral and subcutaneous adipose tissue from 196 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance. Vaspin mRNA expression was only detectable in 23% of the visceral and in 15% of the subcutaneous (SC) adipose tissue samples. Vaspin mRNA expression was not detectable in lean subjects (BMI<25) and was more frequently detected in patients with type 2 diabetes. No significant correlations were found between visceral vaspin gene expression and visceral fat area or SC vaspin expression. However, visceral vaspin expression significantly correlates with BMI, % body fat, and 2 h OGTT plasma glucose. Subcutaneous vaspin mRNA expression is significantly correlated with WHR, fasting plasma insulin concentration, and glucose infusion rate during steady state of an euglycemic-hyperinsulinemic clamp. Multivariate linear regression analysis revealed % body fat as strongest predictor of visceral vaspin and insulin sensitivity as strongest determinant of SC vaspin mRNA expression. In conclusion, our data indicate that induction of human vaspin mRNA expression in adipose tissue is regulated in a fat depot-specific manner and could be associated with parameters of obesity, insulin resistance, and glucose metabolism.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.     

Energy crops as a new bird habitat: utilization of oilseed rape fields by the rare Bluethroat (Luscinia svecica)

As part of the expanding cultivation of renewable primary products, oilseed rape has become a prominent part of the agricultural landscape of many European countries. However, the ecological impacts of increasing cultivation of oilseed rape have received little attention so far. In this study, we provide the first systematic investigation of the suitability of oilseed rape as a habitat for the Bluethroat (Luscinia svecica cyanecula), an endangered and highly specialized bird species. We conducted territorial mapping and a habitat analysis, revealing the distinct habitat requirements of L. s. cyanecula in oilseed rape fields over three breeding seasons. The soil type was identified as the crucial factor for habitat occupancy. It acts as a surrogate for soil moisture and this in turn decisively influences food availability. Ditches can further improve habitat quality, whereas reed is not a necessary structure, although it is a regular feature in the primary habitat of the Bluethroat. Occupied oilseed rape stands were structurally different from the semi-natural reed habitat, but provided similar essential habitat requirements, such as shelter from predation and moist, bare soil. Our results clearly demonstrate the utilization of oilseed rape by a rare and threatened wetland bird species and suggest that this crop may act as a secondary habitat.     

Synthesis and evaluation of substituted hexahydronaphthalenes as novel inhibitors of the Mcl-1/BimBH3 interaction

Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in a broad range of human cancers and plays a critical role in conferring resistance to chemotherapy. In the course of screening a natural product-like library of sesquiterpenoid analogs, we identified substituted hexahydronaphthalenes that showed activity against the Mcl-1/BimBH3 interaction in vitro. Here, we describe the synthesis of a small library of analogs and their biological evaluation. The most potent inhibitor in the series (19) exhibits an IC(50) of 8.3μM by ELISA and disrupts the interaction between endogenously expressed Mcl-1 and Bim in cultured MDA-MB-468 breast cancer cells.     

NAC functions as a modulator of SRP during the early steps of protein targeting to the endoplasmic reticulum

Nascent polypeptide-associated complex (NAC) was initially found to bind to any segment of the nascent chain except signal sequences. In this way, NAC is believed to prevent mistargeting due to binding of signal recognition particle (SRP) to signalless ribosome nascent chain complexes (RNCs). Here we revisit the interplay between NAC and SRP. NAC does not affect SRP function with respect to signalless RNCs; however, NAC does affect SRP function with respect to RNCs targeted to the endoplasmic reticulum (ER). First, early recruitment of SRP to RNCs containing a signal sequence within the ribosomal tunnel is NAC dependent. Second, NAC is able to directly and tightly bind to nascent signal sequences. Third, SRP initially displaces NAC from RNCs; however, when the signal sequence emerges further, trimeric NAC·RNC·SRP complexes form. Fourth, upon docking to the ER membrane NAC remains bound to RNCs, allowing NAC to shield cytosolically exposed nascent chain domains not only before but also during cotranslational translocation. The combined data indicate a functional interplay between NAC and SRP on ER-targeted RNCs, which is based on the ability of the two complexes to bind simultaneously to distinct segments of a single nascent chain.     

The rust fungi (Uredinales) on ferns in South Africa

The rust fungi (Uredinales, basidiomycota) occuring on ferns (Pteridophyta) in South Africa are described, illustrated and keyed out. All species belong to the pucciniastraceous genera Milesina (M. blechni), Uredinopsis (U. pteridis) or to the related uredinial anamorph genus Milesia (M. nervisequa, M. cf. magellanica, M. silvae-knysnae). Milesia silvae-knysnae on Polystichum pungens is new to science; it probably belongs to the teleomorph genus Milesina. Milesina blechni is reported from South Africa for the first time on the new hosts Blechnum punctulatum and Rumohra adiantoides; it has hitherto been known only from the Northern Hemisphere on Blechnum spicant. Rust specimens collected on Asplenium aethiopicum and A. rutifolium were tentatively assigned to Milesia magellanica which has been known so far only from southern Chile. Hyalopsora neocheilanthis, Milesina neoexigua and M. neovogesiaca are proposed as new names for Hyalopsora cheilanthis, Milesia exigua and M. vogesiaca. It is discussed that the pucciniastraceous fern rusts could have reached South Africa either by migration (M. blechni) or by long-distance air dispersal. In the absence of their gametophyte hosts, species of Abies (Pinaceae), the rusts have to propagate in South Africa by urediniospores infecting fern to fern. Taxonomical novelties Milesia silvae-knysnae R. Berndt Milesina neoexigua R. Berndt Milesina neovogesiaca R. Berndt Hyalopsora neocheilanthis R. Berndt