Selenium reduction by a denitrifying consortium

A denitrifying bacterial consortium obtained from the Pullman, Washington wastewater treatment facility was enriched under denitrifying conditions and its ability to reduce selenite and selenate was studied. Replicate experiments at two different experimental conditions were performed. All experiments were performed under electron-acceptor limiting conditions, with acetate as the carbon source and nitrate the electron acceptor. In the first set of experiments, selenite was present, whereas, in the second set, selenate was added. A significant lag period of approximately 150 h was necessary before selenite or selenate reduction was observed. During this lag period, nitrate and nitrite use was observed. Once selenite or selenate reduction had started, nitrate and nitrite reduction was concomitant with selenium species reduction. Trace amounts of selenite were detected during the selenate reduction study. Analysis of the data indicates that, once selenium species reduction was induced, the rate of reduction was proportional to the selenium species concentration and to the biomass concentration. Furthermore, at similar biomass and contaminant concentrations, selenite reduction is approximately four times faster than selenate reduction. Copyright 1999 John Wiley & Sons, Inc.     

The major allergen from birch tree pollen, Bet v 1, binds and permeabilizes membranes

The 159 residue Bet v 1 is the major allergen from birch tree pollen. Its natural function is unknown although it is capable of binding several types of physiologically relevant ligands in a centrally placed cavity in the protein structure. Here we use circular dichroism and fluorescence spectroscopy to show that Bet v 1 binds to DOPC and DOPG phospholipid vesicles in a pH-dependent manner. Binding is facilitated by low pH, negatively charged phospholipids, and high vesicle curvature, indicating that electrostatic interactions and vesicle surface defects are important parameters for binding. Binding is accompanied by major structural rearrangements, involving an increase in alpha-helical structure and a decrease in beta-structure. A bilayer structure per se is not a prerequisite for these rearrangements, since they also occur in the presence of the micelle-forming lysophospholipids lysoMPC and lysoMPG. Two major bound states (A and B) with distinct secondary structure compositions were identified, which predominate in the pH ranges approximately 9.5-6.5 and approximately 5-2.5, respectively. Despite the high content of secondary structure, the A- and B-states are partially unfolded as they unfold noncooperatively in CD thermal scans, in contrast to the native state. In addition, the B-state (but not the A-state) shows intermediate proteolysis-resistance and is able to induce complete leakage of calcein from the vesicles, indicating that this state is partially inserted into and significantly perturbs the bilayer structure. We conclude that Bet v 1 is a membrane binding protein, highlighting a possible biological function of this protein.     

Identification of germline genomic copy number variation in familial pancreatic cancer

Adenocarcinoma of the pancreas is a significant cause of cancer mortality, and up to 10?% of cases appear to be familial. Heritable genomic copy number variants (CNVs) can modulate gene expression and predispose to disease. Here, we identify candidate predisposition genes for familial pancreatic cancer (FPC) by analyzing germline losses or gains present in one or more high-risk patients and absent in a large control group. A total of 120 FPC cases and 1,194 controls were genotyped on the Affymetrix 500K array, and 36 cases and 2,357 controls were genotyped on the Affymetrix 6.0 array. Detection of CNVs was performed by multiple computational algorithms and partially validated by quantitative PCR. We found no significant difference in the germline CNV profiles of cases and controls. A total of 93 non-redundant FPC-specific CNVs (53 losses and 40 gains) were identified in 50 cases, each CNV present in a single individual. FPC-specific CNVs overlapped the coding region of 88 RefSeq genes. Several of these genes have been reported to be differentially expressed and/or affected by copy number alterations in pancreatic adenocarcinoma. Further investigation in high-risk subjects may elucidate the role of one or more of these genes in genetic predisposition to pancreatic cancer.     

Reconstitution of an Active Magnesium Chelatase Enzyme Complex from the bchI, -D, and -H Gene Products of the Green Sulfur Bacterium Chlorobium vibrioforme Expressed in Escherichia coli

Magnesium-protoporphyrin chelatase, the first enzyme unique to the (bacterio)chlorophyll-specific branch of the porphyrin biosynthetic pathway, catalyzes the insertion of Mg²⁺ into protoporphyrin IX. Three genes, designated bchI, -D, and -H, from the strictly anaerobic and obligately phototrophic green sulfur bacterium Chlorobium vibrioforme show a significant level of homology to the magnesium chelatase-encoding genes bchI, -D, and -H and chlI, -D, and -H of Rhodobacter sphaeroides and Synechocystis strain PCC6803, respectively. These three genes were expressed in Escherichia coli; the subsequent purification of overproduced BchI and -H proteins on an Ni²⁺-agarose affinity column and denaturation of insoluble BchD protein in 6 M urea were required for reconstitution of Mg-chelatase activity in vitro. This work therefore establishes that the magnesium chelatase of C. vibrioforme is similar to the magnesium chelatases of the distantly related bacteria R. sphaeroides and Synechocystis strain PCC6803 with respect to number of subunits and ATP requirement. In addition, reconstitution of an active heterologous magnesium chelatase enzyme complex was obtained by combining the C. vibrioforme BchI and -D proteins and the Synechocystis strain PCC6803 ChlH protein. Furthermore, two versions, with respect to the N-terminal start of the bchI gene product, were expressed in E. coli, yielding ca. 38- and ca. 42-kDa versions of the BchI protein, both of which proved to be active. Western blot analysis of these proteins indicated that two forms of BchI, corresponding to the 38- and the 42-kDa expressed proteins, are also present in C. vibrioforme.     

Biosynthesis and metabolic engineering of glucosinolates

Summary. Glucosinolates are amino acid-derived natural plant products found throughout the Capparales order. Glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi. The conversion of amino acids to aldoximes is a key step in glucosinolate biosynthesis. This step is catalyzed by cytochromes P450 from the CYP79 family. The post-aldoxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional group and low substrate-specificity for the side chain. Therefore, we have been able to metabolically engineer new glucosinolate profiles into Arabidopsis by altering the levels of endogenous CYP79s and by introducing exogenous CYP79s. The approach has great potential for design of metabolically engineered plants with improved pest resistance and increased nutritional value. Received December 7, 2001 Accepted December 21, 2001     

Segregation of saturated chain lipids in pulmonary surfactant films and bilayers

The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domain's shape compared with that observed between 37 degrees C to 20 degrees C, suggesting the coexistence of two ordered but differently organized lipid phases on the bilayer. Epifluorescence microscopy studies of BLES monomolecular films doped with small amounts of fluorescent lipids showed the appearance and growth of dark domains (liquid condensed) dispersed in a fluorescent phase (liquid expanded) with shapes and sizes similar to those observed in BLES giant unilamelar vesicles. Our study suggests that bovine surfactant lipids can organize into discrete phases in monolayers or bilayers with equivalent temperature dependencies and may occur at physiological temperatures and surface pressures equivalent to those at the lung interface.     

Dynamics of a microbial community associated with manure hot spots as revealed by phospholipid fatty acid analyses.

Microbial community dynamics associated with manure hot spots were studied by using a model system consisting of a gel-stabilized mixture of soil and manure, placed between layers of soil, during a 3-week incubation period. The microbial biomass, measured as the total amount of phospholipid fatty acids (PLFA), had doubled within a 2-mm distance from the soil-manure interface after 3 days. Principal-component analyses demonstrated that this increase was accompanied by reproducible changes in the composition of PLFA, indicating changes in the microbial community structure. The effect of the manure was strongest in the 2-mm-thick soil layer closest to the interface, in which the PLFA composition was statistically significantly different (P < 0.05) from that of the unaffected soil layers throughout the incubation period. An effect was also observed in the soil layer 2 to 4 mm from the interface. The changes in microbial biomass and community structure were mainly attributed to the diffusion of dissolved organic carbon from the manure. During the initial period of microbial growth, PLFA, which were already more abundant in the manure than in the soil, increased in the manure core and in the 2-mm soil layer closest to the interface. After day 3, the PLFA composition of these layers gradually became more similar to that of the soil. The dynamics of individual PLFA suggested that both taxonomic and physiological changes occurred during growth. Examples of the latter were decreases in the ratios of 16:1 omega 7t to 16:1 omega 7c and of cyclopropyl fatty acids to their respective precursors, indicating a more active bacterial community. An inverse relationship between bacterial PLFA and the eucaryotic 20:4 PLFA (arachidonic acid) suggested that grazing was important.     

Studien zur vergleichenden und allgemeinen Mechanik des Tierkörpers

Ohne Zusammenfassung     

LRIG1 is a novel negative regulator of the Met receptor and opposes Met and Her2 synergy

The Met receptor tyrosine kinase regulates a complex array of cellular behaviors collectively known as "invasive growth." While essential for normal development and wound repair, this program is frequently co-opted by tumors to promote their own growth, motility, and invasion. Met is overexpressed in a variety of human tumors, and this aberrant expression correlates with poor patient prognosis. Previous studies indicate that Met receptor levels are governed in part by cbl-mediated ubiquitination and degradation, and uncoupling of Met from cbl-mediated ubiquitination promotes its transforming activity. Here we describe a novel mechanism for Met degradation. We find that the Met receptor interacts with the transmembrane protein LRIG1 independent of hepatocyte growth factor (HGF) stimulation and that LRIG1 destabilizes the Met receptor in a cbl-independent manner. Overexpression of LRIG1 destabilizes endogenous Met receptor in breast cancer cells and impairs their ability to respond to HGF. LRIG1 knockdown increases Met receptor half-life, indicating that it plays an essential role in Met degradation. Finally, LRIG1 opposes Met synergy with the ErbB2/Her2 receptor tyrosine kinase in driving cellular invasion. We conclude that LRIG1 is a novel suppressor of Met function, serving to regulate cellular receptor levels by promoting Met degradation in a ligand- and cbl-independent manner.     

Alpha-LNA (alpha-D-configured locked nucleic acid)

Two pyrimidine alpha-LNA nucleoside monomers have been synthesised and incorporated into alpha-configured oligonucleotides. A fully modified mixed alpha-LNA sequence displays unprecedented parallel stranded hybridisation with complementary RNA and a remarkable selectivity for RNA over DNA. Modelling shows alpha-LNA:RNA to form an extended duplex with a very broad major groove.