Photoaffinity labeling of DNA-dependent RNA polymerase from Escherichia coli with 8-azidoadenosine 5'-triphosphate

A photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azidoadenosine 5'-triphosphate (8-N3ATP), has been used to elucidate the role of the various subunits involved in forming the active site of Escherichia coli DNA-dependent RNA polymerase. 8-N3ATP was found to be a competitive inhibitor of the enzyme with respect to the incorporation of ATP with Ki = 42 microM, while uridine 5'-triphosphate (UTP) incorporation was not affected. UV irradiation of the reaction mixture containing RNA polymerase and [gamma-32P]-8-N3ATP induced covalent incorporation of radioactive label into the enzyme. Analysis by gel filtration and nitrocellulose filter binding indicated specific binding. Subunit analysis by sodium dodecyl sulfate and sodium tetradecyl sulfate gel electrophoresis and autoradiography of the labeled enzyme showed that the major incorporation of radioactive label was in beta' and sigma, with minor incorporation in beta and alpha. The same pattern was observed in both the presence and absence of poly[d(A-T)] and poly[d(A-T)] plus ApU. Incorporation of radioactive label in all bands was significantly reduced by 100-150 microM ATP, while 100-200 microM UTP did not show a noticeable effect. Our results indicate major involvement of the beta' and sigma subunits in the active site of RNA polymerase. The observation of a small extent of labeling of the beta and alpha subunits, which was prevented by saturating levels of ATP, suggests that these subunits are in close proximity to the catalytic site.     

Habitat and distribution of perforatella rubiginosa (Gastropoda,Pulmonata) in the freshwater tidal region of the Scheldt estuary,Belgium

The amphibious land-molluse,Perforatella rubiginosa, is a common, wide-spread and characteristic inhabitant of the freshwater tidal region of the river Scheldt and its tributaries in NW Belgium. The species occupies a rather narrow zone in the uppermost intertidal area, which is characterized by large amounts of tidal drift covering the bottom, and usually by a luxuriant vegetation of tall, nitrophilous herbs, often withUrtica dioica dominant. Other characteristic molluscs of this zone areCarychium minimum, Oxyloma pfeifferi, O. sarsi, Deroceras laeve, Vitrea crystallina andZonitoides nitidus, locally alsoNesovitrea hammonis. Communication nr. 143 of the Delta Institute for Hydrobiological Research, Yerseke, The Netherlands     

Detection and identification of FaeC as a minor component of K88 fibriilae of Escherichia coli

A tribrid gene containing ompF, faeC, and lacZ sequences was constructed by subcloning a large central segment of the K88ab gene encoding the fibrillar subunit-like protein FaeC into the open reading frame expression vector pORF2. The resulting tribrid protein was isolated and used to raise antibodies against the FaeC protein. These antibodies were then used for the detection and subcellular localization of the FaeC protein in Escherichia coli harbouring the K88ab-encoding plasmid pFM205 or mutant derivatives. Immunoblotting of subcellular fractions and of purified fibrillae, and agglutination experiments using whole cells revealed that the FaeC protein is present in the periplasm and as a minor component in the K88ab fibrillae. FaeC was also detected in purified K88ac and K88ad fibrillae. Immunoelectron microscopy confirmed the presence of FaeC in K88ab fibrillae, particularly at the tips of the longer fibrillae.     

Gut microbiota composition is associated with environmental landscape in honey bees

There is growing recognition that the gut microbial community regulates a wide variety of important functions in its animal hosts, including host health. However, the complex interactions between gut microbes and environment are still unclear. Honey bees are ecologically and economically important pollinators that host a core gut microbial community that is thought to be constant across populations. Here, we examined whether the composition of the gut microbial community of honey bees is affected by the environmental landscape the bees are exposed to. We placed honey bee colonies reared under identical conditions in two main landscape types for 6 weeks: either oilseed rape farmland or agricultural farmland distant to fields of flowering oilseed rape. The gut bacterial communities of adult bees from the colonies were then characterized and compared based on amplicon sequencing of the 16S rRNA gene. While previous studies have delineated a characteristic core set of bacteria inhabiting the honey bee gut, our results suggest that the broad environment that bees are exposed to has some influence on the relative abundance of some members of that microbial community. This includes known dominant taxa thought to have functions in nutrition and health. Our results provide evidence for an influence of landscape exposure on honey bee microbial community and highlight the potential effect of exposure to different environmental parameters, such as forage type and neonicotinoid pesticides, on key honey bee gut bacteria. This work emphasizes the complexity of the relationship between the host, its gut bacteria, and the environment and identifies target microbial taxa for functional analyses.     

A Blind Spot in Confocal Reflection Microscopy: The Dependence of Fiber Brightness on Fiber Orientation in Imaging Biopolymer Networks

We investigate the dependence of fiber brightness on three-dimensional fiber orientation when imaging biopolymer networks with confocal reflection microscopy (CRM) and confocal fluorescence microscopy (CFM). We compare image data of fluorescently labeled type I collagen networks concurrently acquired using each imaging modality. For CRM, fiber brightness decreases for more vertically oriented fibers, leaving fibers above ∼50° from the imaging plane entirely undetected. As a result, the three-dimensional network structure appears aligned with the imaging plane. In contrast, CFM data exhibit little variation of fiber brightness with fiber angle, thus revealing an isotropic collagen network. Consequently, we find that CFM detects almost twice as many fibers as are visible with CRM, thereby yielding more complete structural information for three-dimensional fiber networks. We offer a simple explanation that predicts the detected fiber brightness as a function of fiber orientation in CRM.     

Molybdenum cofactor from the cytoplasmic membrane of Proteus mirabilis

Molybdenum cofactor was extracted from membranes of Proteus mirabilis by three methods: acidification, heat treatment and heat treatment in the presence of sodium-dodecylsulphate (SDS). Extracts prepared by the latter method contained the highest concentration of molybdenum cofactor. In these extracts molybdenum cofactor was present in a low molecular weight form. It could not penetrate an YM-2 membrane during ultrafiltration suggesting a molecular weight above 1000. During aerobic incubation of cofactor extracts from membranes at least four fluorescent species were formed as observed in a reversed-phase high performance liquid chromatography (HPLC) system. The species in the first peak was inhomogeneous while the species in the others seem to be homogenous. In water, all fluorescent products had an excitation maximum at 380 nm and an emission maximum at 455 nm. Their absorption spectra showed maxima at around 270 nm and 400 nm. Fluorescent compounds present in the first peak could penetrate an YM-2 membrane during ultrafiltration, whereas the compounds in the other peaks hardly did. Using xanthine oxidase from milk as source of molybdenum cofactor apparently identical cofactor species were found. Cytoplasmic nor membrane extracts of the chlorate resistant mutant chl S 556 of P. mirabilis could complement nitrate reductase of Neurospora crassa nit-1 in the presence of 20 mM molybdate. However, fluorescent species with identical properties as found for the wild-type were formed during aerobic incubation of extracts from membranes of this mutant.Non-common Abbreviations HPLC high performance liquid chromatography - I.D. internal diameter - SDS sodium dodecyl sulphate     

The case for Survivin as mitotic regulator

Survivin has been proposed to inhibit apoptosis and to regulate cell division. However, controversy still exists as to whether Survivin can indeed execute these distinct functions and if Survivin somehow coordinates apoptosis and (abnormal) cell division. Recent evidence has demonstrated that Survivin acts as a subunit of the chromosomal passenger complex, which is essential for proper chromosome segregation and cytokinesis. Within this complex, the mitotic kinase Aurora B acts as the enzymatic core, whereas Survivin dictates chromosomal passenger complex localization. This function of Survivin appears to be conserved throughout evolution. Although these findings do not exclude a role for Survivin as apoptosis inhibitor, they make a very strong case for Survivin as mitotic regulator.