In situ exposure to low herbicide concentrations affects microbial population composition and catabolic gene frequency in an aerobic shallow aquifer

The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 micro g l(-1)) changes the microbial community composition. Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations, Pseudomonas bacteria, and specific phenoxy acid degraders. Both culture-dependent and culture-independent methods were applied. The abundance of microbial phenoxy acid degraders (10(0) to 10(4) g(-1) sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments. Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes tfdA and tfdB (10(2) to 10(3) gene copies g(-1) sediment) were only detected in sediments from contaminated areas of the aquifer. PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of tfd genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders. The number of Pseudomonas bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid. Furthermore, high numbers of CFU compared to direct counting of 4',6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments. The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer.     

Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy

Aminoacyl-tRNAs (aa-tRNAs) are delivered to the ribosome as part of the ternary complex of aa-tRNA, elongation factor Tu (EF-Tu) and GTP. Here, we present a cryo-electron microscopy (cryo-EM) study, at a resolution of approximately 9 A, showing that during the incorporation of the aa-tRNA into the 70S ribosome of Escherichia coli, the flexibility of aa-tRNA allows the initial codon recognition and its accommodation into the ribosomal A site. In addition, a conformational change observed in the GTPase-associated center (GAC) of the ribosomal 50S subunit may provide the mechanism by which the ribosome promotes a relative movement of the aa-tRNA with respect to EF-Tu. This relative rearrangement seems to facilitate codon recognition by the incoming aa-tRNA, and to provide the codon-anticodon recognition-dependent signal for the GTPase activity of EF-Tu. From these new findings we propose a mechanism that can explain the sequence of events during the decoding of mRNA on the ribosome.     

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.     

Bi- and tricyclic nucleoside derivatives restricted in S-type conformations and obtained by RCM-reactions

Ring-closing metathesis (RCM) is applied as a new and powerful technology in the construction of nucleoside analogues that are conformationally restricted in S-type conformations due to additional 3',4'- and/or 3',5'-linkages.     

Estrogenic effect of propylparaben (propylhydroxybenzoate) in rainbow trout Oncorhynchus mykiss after exposure via food and water

The estrogenic effect of propylparaben was investigated in a rainbow trout Oncorhynchus mykiss test system. Propylparaben was administered orally to sexually immature rainbow trout every second day for up to 10 days in doses between 7 and 1830 mg kg(-1) 2 d(-1) and in the water at 50 and 225 microg l(-1) for 12 days. Plasma vitellogenin was measured before and during the exposures and the concentrations of propylparaben in liver and muscle were determined at the end of experiments. Increases in average plasma vitellogenin levels were seen at oral exposure to 33 mg propylparabenkg(-1) 2 d(-1); the most sensitive fish responded to 7 mg kg(-1). The ED(50) values for increase in vitellogenin synthesis were 35, 31 and 22 mg kg(-1) 2 d(-1) at day 3, 6 and 11, respectively. Exposure to 225 microg propylparabenl(-1) increased vitellogenin synthesis, but exposure to 50 microg l(-1) did not. Propylparaben showed little tendency to bioaccumulation in rainbow trout; less than 1 per thousand of the total amount of propylparaben administered orally at 1830 mg kg(-1) 2 d(-1) over the 10-d experimental period was retained in muscle and liver 24 h after the end of the experiment. Exposure to 225 microg propylparabenl(-1) for 12 d led to concentrations of 6700 and 870 microg propylparabenkg(-1) liver and muscle, respectively. Half lives for propylparaben were 8.6 h in liver and 1.5 h in muscle.     

Sensory and biomechanical responses to ramp-controlled distension of the human duodenum

The aim of this study was to develop a new method for investigation of the relationship among the mechanical stimulus, the biomechanical properties, and the visceral perception evoked by volume/ramp-controlled distension in the human duodenum in vivo. An impedance planimetric probe for balloon distension was placed in the third part of the duodenum in seven healthy volunteers. Distension of the duodenum was done at infusion rates of 10, 25, and 50 ml/min. The pump was reversed when level 7 was reached on a visual analog scale ranging from 0 to 10. Distensions were done with and without the administration of the antimuscarinic drug butylscopolamine. The total circumferential tension (T(total)) and the passive circumferential tension (T(passive)) were determined from the distension tests without and with the administration of butylscopolamine, respectively. T(total) and T(passive) showed an exponential behavior as a function of strain (a measure of deformation). The active circumferential tension (T(active)) was computed as T(total)-T(passive) and showed a bell-shaped behavior as a function of strain. At low distension intensities, the intensity of sensation at 10 ml/min was significantly higher than that obtained at 25 and 50 ml/min. The coefficient of variation at the pain threshold for circumferential strain (average 4.34) was closer to zero compared with those for volume (8.72), pressure (31.22), and circumferential tension (31.55). This suggests that the mechanoreceptors in the gastrointestinal wall depend primarily on circumferential strain. The stimulus-response functions provided evidence for the existence of low- and high-threshold mechanoreceptors in the human duodenum. Furthermore, the data suggest that high-threshold receptors are nonadapting.     

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.     

Phenothiazines alter plasma membrane properties and sensitize cancer cells to injury by inhibiting annexin-mediated repair

Repair of damaged plasma membrane in eukaryotic cells is largely dependent on the binding of annexin repair proteins to phospholipids. Changing the biophysical properties of the plasma membrane may provide means to compromise annexin-mediated repair and sensitize cells to injury. Since, cancer cells experience heightened membrane stress and are more dependent on efficient plasma membrane repair, inhibiting repair may provide approaches to sensitize cancer cells to plasma membrane damage and cell death. Here, we show that derivatives of phenothiazines, which have widespread use in the fields of psychiatry and allergy treatment, strongly sensitize cancer cells to mechanical-, chemical-, and heat-induced injury by inhibiting annexin-mediated plasma membrane repair. Using a combination of cell biology, biophysics, and computer simulations, we show that trifluoperazine acts by thinning the membrane bilayer, making it more fragile and prone to ruptures. Secondly, it decreases annexin binding by compromising the lateral diffusion of phosphatidylserine, inhibiting the ability of annexins to curve and shape membranes, which is essential for their function in plasma membrane repair. Our results reveal a novel avenue to target cancer cells by compromising plasma membrane repair in combination with noninvasive approaches that induce membrane injuries.     

The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers

Transformation and cancer growth are regulated by the coordinate actions of oncogenes and tumor suppressors. Here, we show that the novel E3 ubiquitin ligase HACE1 is frequently downregulated in human tumors and maps to a region of chromosome 6q21 implicated in multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late-onset cancer. A second hit from either environmental triggers or genetic heterozygosity of another tumor suppressor, p53, markedly increased tumor incidence in a Hace1-deficient background. Re-expression of HACE1 in human tumor cells directly abrogates in vitro and in vivo tumor growth, whereas downregulation of HACE1 via siRNA allows non-tumorigenic human cells to form tumors in vivo. Mechanistically, the tumor-suppressor function of HACE1 is dependent on its E3 ligase activity and HACE1 controls adhesion-dependent growth and cell cycle progression during cell stress through degradation of cyclin D1. Thus, HACE1 is a candidate chromosome 6q21 tumor-suppressor gene involved in multiple cancers.     

Differential effect of HOE642 on two separate monovalent cation transporters in the human red cell membrane.

Residual K(+) fluxes in red blood cells can be stimulated in conditions of low ionic strength. Previous studies have identified both the non-selective, voltage-dependent cation (NSVDC) channel and the K(+)(Na(+))/H(+) exchanger as candidate pathways mediating this effect, although it is possible that these pathways represent different modes of operation of a single system. In the present study the effects of HOE642, recently characterised as an inhibitor of the K(+)(Na(+))/H(+) exchanger, on NSVDC has been determined to clarify this question. Radioisotope flux measurements and conductance determinations showed that HOE642 exerted differential effects on the NSVDC channel and the K(+)(Na(+))/H(+) exchanger, confirming that the salt loss observed in low ionic strength solutions represents contributions from at least two independent ion transport pathways. The findings are discussed in the context of red blood cell apoptosis (eryptosis) and disease.