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Circadian Rhythms in the Thermophilic Cyanobacterium Thermosynechococcus elongatus: Compensation of Period Length over a Wide Temperature Range. Kiyoshi Onai, 2004.Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies . We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium T . elongatus BP-1 that uses a bacterial luciferase gene set (Xl luxAB) derived from Xenorhabdus luminescens as a bioluminescence reporter gene . A promoter region of the psbA1 gene of T . elongatus was fused to the Xl luxAB gene set and inserted into a specific targeting site in the genome of T . elongatus . The bioluminescence from the cells of the psbA1-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes . The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature . The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60°C) . Theses results indicate that T . elongatus has the circadian clock that is widely temperature compensated . Antimalarial Quinolines and Artemisinin Inhibit Endocytosis in Plasmodium falciparum. Heinrich C. Hoppe, 2004.Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake . Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole . In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum . By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite . During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers . By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40% . Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion . This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin . These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites . Regulation of Purine Hydroxylase and Xanthine Dehydrogenase from Clostridium purinolyticum in Response to Purines, Selenium, and Molybdenum. William T. Self, 2002.The discovery that two distinct enzyme catalysts, purine hydroxylase (PH) and xanthine dehydrogenase (XDH), are required for the overall conversion of hypoxanthine to uric acid by Clostridium purinolyticum was unexpected . In this reaction sequence, hypoxanthine is hydroxylated to xanthine by PH and then xanthine is hydroxylated to uric acid by XDH . PH and XDH, which contain a labile selenium cofactor in addition to a molybdenum cofactor, flavin adenine dinucleotide, and FeS centers, were purified and partially characterized as reported previously . In the present study, the activities of these two enzymes were measured in cells grown in media containing various concentrations of selenite, molybdate, and various purine substrates . The levels of PH protein in extracts were determined by immunoblot assay . The amount of PH protein, as well as the specific activities of PH and XDH, increased when either selenite or molybdate was added to the culture medium . PH levels were highest in the cells cultured in the presence of either adenine or purine . XDH activity increased dramatically in cells grown with either xanthine or uric acid . The apparent increases in protein levels and activities of PH and XDH in response to selenium, molybdenum, and purine substrates demonstrate that these enzymes are tightly regulated in response to these nutrients . Effect of Particles on the Recovery of Cryptosporidium Oocysts from Source Water Samples of Various Turbidities. Yao Yu Feng, 2003.Cryptosporidium parvum can be found in both source and drinking water and has been reported to cause serious waterborne outbreaks which threaten public health safety . The U.S . Environmental Protection Agency has developed method 1622 for detection of Cryptosporidium oocysts present in water . Method 1622 involves four key processing steps: filtration, immunomagnetic separation (IMS), fluorescent-antibody (FA) staining, and microscopic evaluation . The individual performance of each of these four steps was evaluated in this study . We found that the levels of recovery of C . parvum oocysts at the IMS-FA and FA staining stages were high, averaging more than 95% . In contrast, the level of recovery declined significantly, to 14.4%, when the filtration step was incorporated with tap water as a spiking medium . This observation suggested that a significant fraction of C . parvum oocysts was lost during the filtration step . When C . parvum oocysts were spiked into reclaimed water, tap water, microfiltration filtrate, and reservoir water, the highest mean level of recovery of (85.0% ± 5.2% [mean ± standard deviation]) was obtained for the relatively turbid reservoir water . Further studies indicated that it was the suspended particles present in the reservoir water that contributed to the enhanced C . parvum oocyst recovery . The levels of C . parvum oocyst recovery from spiked reservoir water with different turbidities indicated that particle size and concentration could affect oocyst recovery . Similar observations were also made when silica particles of different sizes and masses were added to seeded tap water . The optimal particle size was determined to be in the range from 5 to 40 µm, and the corresponding optimal concentration of suspended particles was 1.42 g for 10 liters of tap water . Occurrence of Escherichia coli and Enterococci in Cladophora (Chlorophyta) in Nearshore Water and Beach Sand of Lake Michigan. Richard L. Whitman, 2003.Each summer, the nuisance green alga Cladophora (mostly Cladophora glomerata) amasses along Lake Michigan beaches, creating nearshore anoxia and unsightly, malodorous mats that can attract problem animals and detract from visitor enjoyment . Traditionally, elevated counts of Escherichia coli are presumed to indicate the presence of sewage, mostly derived from nearby point sources . The relationship between fecal indicator bacteria and Cladophora remains essentially unstudied . This investigation describes the local and regional density of Escherichia coli and enterococci in Cladophora mats along beaches in the four states (Wisconsin, Illinois, Indiana, and Michigan) bordering Lake Michigan . Samples of Cladophora strands collected from 10 beaches (n = 41) were assayed for concentrations of E . coli and enterococci during the summer of 2002 . Both E . coli and enterococci were ubiquitous (up to 97% occurrence), with overall log mean densities (± standard errors) of 5.3 (± 4.8) and 4.8 (± 4.5) per g (dry weight) . E . coli and enterococci were strongly correlated in southern Lake Michigan beaches (P < 0.001, R2 = 0.73, n = 17) but not in northern beaches (P = 0.892, n = 16) . Both E . coli and enterococci survived for over 6 months in sun-dried Cladophora mats stored at 4°C; the residual bacteria in the dried alga readily grew upon rehydration . These findings suggest that Cladophora amassing along the beaches of Lake Michigan may be an important environmental source of indicator bacteria and call into question the reliability of E . coli and enterococci as indicators of water quality for freshwater recreational beaches .
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