Measured and predicted environmental concentrations of carbamazepine, diclofenac, and metoprolol in small and medium rivers in northern Germany.

This study evaluated the impact of secondary municipal effluent discharge on carbamazepine, diclofenac, and metoprolol concentrations in small and medium rivers in northern Germany and compared the measured environmental concentrations (MECs) to the predicted environmental concentrations (PECs) calculated with four well-established models. During a 1-year sampling period, secondary effluent grab samples were collected at four wastewater treatment plants (WWTPs) together with grab samples from the receiving waters upstream and downstream from the wastewater discharge points. The carbamazepine, diclofenac, and metoprolol concentrations were analyzed with high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS-MS) after solid phase extraction. In the secondary effluents, 84-790 ng/L carbamazepine, 395-2100 ng/L diclofenac, and 745-5000 ng/L metoprolol were detected. The carbamazepine, diclofenac, and metoprolol concentrations analyzed in the rivers downstream from the secondary effluent discharge sites ranged from <5 to 68, 370, and 520 ng/L, respectively. Most of the downstream pharmaceutical concentrations were markedly higher than the corresponding upstream concentrations. The impact of wastewater discharge on the MECs in rivers downstream from the WWTPs was clearly demonstrated, but the correlations of the MECs with dilution factors were poor. The smallest rivers exhibited the largest maximum MECs and the widest ranges of MECs downstream from the wastewater discharge point. Three of the four tested models were conservative, as they showed higher PECs than the MECs in the rivers downstream from the WWTPs. However, the most detailed model underestimated the diclofenac concentrations.

Inadequacy of carbamazepine-spiked model wastewaters for testing photocatalysis efficiency.

The study was performed in order to clarify whether carbamazepine-spiked solutions used as model wastewaters are suitable for the assessment of carbamazepine removal from real secondary municipal effluents by photocatalytic oxidation in the presence and absence of activated carbon. Therefore, carbamazepine (10 mg L(-1)) was dissolved in deionized water or in secondary municipal effluent. Photocatalytic oxidation of these model wastewaters was carried out with TiO2 "P25" (100 mg L(-1)) and UV-A lamps in the absence and in the presence of 20 mg L(-1) powdered activated carbon (PAC). Carbamazepine was analyzed photometrically. In deionized water at pH 5.5, carbamazepine was nearly completely removed with a UV dose of 6.48 kJ L(-1). A similar efficiency of photocatalytic oxidation of carbamazepine added to secondary effluent was observed when the suspension pH was 2.7, while at pH 8 and 10.6, carbamazepine removal from spiked secondary effluent with the same UV dose was only 40 and 60%, respectively. Although PAC addition resulted in an initial adsorptive carbamazepine reduction of 20 to 35% from the model wastewaters, it did not lead to markedly enhanced carbamazepine removal in the subsequent photocatalysis phase. During photocatalytic oxidation of unspiked secondary effluent (initial carbamazepine concentration: 133 ng L(-1)) at pH 7.3 with and without PAC, carbamazepine concentrations were analyzed by HPLC/MS/MS. While PAC addition resulted in the adsorption of about 90% of the initial carbamazepine, photocatalysis did not lead to any carbamazepine removal at all. This indicates that the experiments with spiked model wastewaters ­ even in a secondary effluent matrix ­ are absolutely inadequate for predicting photocatalytic carbamazepine removal under real conditions.

Natural variation of the amino-terminal glutamine-rich domain in Drosophila argonaute2 is not associated with developmental defects.

The Drosophila argonaute2 (ago2) gene plays a major role in siRNA mediated RNA silencing pathways. Unlike mammalian Argonaute proteins, the Drosophila protein has an unusual amino-terminal domain made up largely of multiple copies of glutamine-rich repeats (GRRs). We report here that the ago2 locus produces an alternative transcript that encodes a putative short isoform without this amino-terminal domain. Several ago2 mutations previously reported to be null alleles only abolish expression of the long, GRR-containing isoform. Analysis of drop out (dop) mutations had previously suggested that variations in GRR copy number result in defects in RNAi and embryonic development. However, we find that dop mutations genetically complement transcript-null alleles of ago2 and that ago2 alleles with variant GRR copy numbers support normal development. In addition, we show that the assembly of the central RNAi machinery, the RISC (RNA induced silencing complex), is unimpaired in embryos when GRR copy number is altered. In fact, we find that GRR copy number is highly variable in natural D. melanogaster populations as well as in laboratory strains. Finally, while many other insects share an extensive, glutamine-rich Ago2 amino-terminal domain, its primary sequence varies drastically between species. Our data indicate that GRR variation does not modulate an essential function of Ago2 and that the amino-terminal domain of Ago2 is subject to rapid evolution.

The C-terminal region of the meiosis-specific protein kinase Ime2 mediates protein instability and is required for normal spore formation in budding yeast.

The cyclin-dependent kinase Cdk1 and the related kinase Ime2 act in concert to trigger progression of the meiotic cell cycle in the yeast Saccharomyces cerevisiae. These kinases share several functions and substrates during meiosis, but their regulation seems to be clearly different. In contrast to Cdk1, no cyclin seems to be involved in the regulation of Ime2 activity. Ime2 is a highly unstable protein, and we aimed to elucidate the relevance of Ime2 instability. We first determined the sequence elements required for Ime2 instability by constructing a set of deletions in the IME2 gene. None of the small deletions in Ime2 affected its instability, but deletion of a 241 amino acid C-terminal region resulted in a highly stabilized protein. Thus, the C-terminal domain of Ime2 is important for mediating protein instability. The stabilized, truncated Ime2 protein is highly active in vivo. Replacement of the IME2 gene with the truncated IME2DeltaC241 in diploid strains did not interfere with meiotic nuclear divisions, but caused abnormalities in spore formation, as manifested by the appearance of many asci with a reduced spore number such as triads and dyads. The truncated Ime2 caused a reduction of spore number in a dominant manner. We conclude that downregulation of Ime2 kinase activity mediated by the C-terminal domain is required for the efficient production of normal four-spore asci. Our data suggest a role for Ime2 in spore number control in S. cerevisiae.

Overlapping functions of argonaute proteins in patterning and morphogenesis of Drosophila embryos.

Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs) of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning.

Enzyme production by the nematode-trapping fungus, Duddingtonia flagrans.

Duddingtonia flagrans degrades peptides, proteins, starch, pectin, lipase, lecithin and oils when grown on agar medium. Serine proteases with optimal activity at pH 8.5 to 10.5 were produced when it was grown in submerged culture. It also produced phospholipase C with optimal activity at pH 8.5, lipases with high activity at pH 3.5 and at 7.5 to 8.5 and pectin-degrading enzymes with pH optima of 3 and 8. The pH of the culture medium affected the types of lipases and pectin degrading enzymes produced but not the proteases or phospholipase C.