Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

Electromagnetic field exposure and health among RF plastic sealer operators

Operators of RF plastic sealers (RF operators) are an occupational category highly exposed to radiofrequency electromagnetic fields. The aim of the present study was to make an appropriate exposure assessment of RF welding and examine the health status of the operators. In total, 35 RF operators and 37 controls were included. The leakage fields (electric and magnetic field strength) were measured, as well as induced and contact current. Information about welding time and productivity was used to calculate time integrated exposure. A neurophysiological examination and 24 h ECG were also carried out. The participants also had to answer a questionnaire about subjective symptoms. The measurements showed that RF operators were exposed to rather intense electric and magnetic fields. The mean values of the calculated 6 min, spatially averaged E and H field strengths, in line with ICNIRP reference levels, are 107 V/m and 0.24 A/m, respectively. The maximum measured field strengths were 2 kV/m and 1.5 A/m, respectively. The induced current in ankles and wrists varied, depending on the work situation, with a mean value of 101 mA and a maximum measured value of 1 A. In total, 11 out of 46 measured RF plastic sealers exceeded the ICNIRP reference levels. RF operators, especially the ready made clothing workers had a slightly disturbed two-point discrimination ability compared to a control group. A nonsignificant difference between RF operators and controls was found in the prevalence of subjective symptoms, but the time integrated exposure parameters seem to be of importance to the prevalence of some subjective symptoms: fatigue, headaches, and warmth sensations in the hands. Further, RF operators had a significantly lower heart rate (24 h registration) and more episodes of bradycardia compared to controls.     

Discovery of a potent, selective and orally active canine COX-2 inhibitor, 2-(3-difluoromethyl-5-phenyl-pyrazol-1-yl)-5-methanesulfonyl-pyridine

Structure-activity relationship (SAR) studies of 2-[3-di(and tri)fluoromethyl-5-arylpyrazol-1-yl]-5-methanesulfonylpyridine derivatives for canine COX enzymes are described. This led to the identification of 12a as a lead candidate for further progression. The in vitro and in vivo activity of 12a for the canine COX-2 enzyme as well as its in vivo efficacy and pharmacokinetic properties in dog are highlighted.     

Computer simulations improve university instructional laboratories

Laboratory classes are commonplace and essential in biology departments but can sometimes be cumbersome, unreliable, and a drain on time and resources. As university intakes increase, pressure on budgets and staff time can often lead to reduction in practical class provision. Frequently, the ability to use laboratory equipment, mix solutions, and manipulate test animals are essential learning outcomes, and "wet" laboratory classes are thus appropriate. In others, however, interpretation and manipulation of the data are the primary learning outcomes, and here, computer-based simulations can provide a cheaper, easier, and less time- and labor-intensive alternative. We report the evaluation of two computer-based simulations of practical exercises: the first in chromosome analysis, the second in bioinformatics. Simulations can provide significant time savings to students (by a factor of four in our first case study) without affecting learning, as measured by performance in assessment. Moreover, under certain circumstances, performance can be improved by the use of simulations (by 7% in our second case study). We concluded that the introduction of these simulations can significantly enhance student learning where consideration of the learning outcomes indicates that it might be appropriate. In addition, they can offer significant benefits to teaching staff.     

Residency effects in animal contests

The question of why territorial residents usually win asymmetrical owner-intruder contests is critical to our understanding of animal contest evolution. Game theory suggests that, under certain conditions, residency could be used as an arbitrary means of contest settlement in a manner analogous to tossing a coin. Key empirical support for this idea is provided by a study on the speckled wood butterfly (Pararge aegeria); however, this result has proven controversial. We show conclusively that residency does not serve as an arbitrary cue for contest settlement in this species. By means of a series of manipulative experiments, conducted on two phenotypically divergent populations of P. aegeria, we also rule out the recently presented alternative that contests are settled due to resource-correlated asymmetries in thoracic temperature. Our results instead suggest that more intrinsically aggressive males accumulate as residents and continue to win due to the self-reinforcing effect of prior winning experience. Truly arbitrary contest settlement may be rare or non-existent in the wild.     

A new purple sulfur bacterium isolated from a littoral microbial mat,<Emphasis Type="Italic"> Thiorhodococcus drewsii</Emphasis> sp. nov.

A new strain of purple sulfur bacterium was isolated from a marine microbial mat sampled in Great Sippewissett Salt Marsh at the Atlantic coast (Woods Hole, Mass., USA). Single cells of strain AZ1 were coccus-shaped, highly motile by means of a single flagellum, and did not contain gas vesicles. Intracellular membranes were of the vesicular type. However, additional concentric membrane structures were present. The photosynthetic pigments were bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series, with rhodopin as the dominant carotenoid. Hydrogen sulfide (up to 11 mM), sulfur, thiosulfate, and molecular hydrogen were used as electron donors during anaerobic phototrophic growth. During growth on sulfide, elemental sulfur globules were transiently stored inside the cells. Strain AZ1 is much more versatile than most other Chromatiaceae with respect to electron donor and organic substrates. In the presence of CO(2), it is capable of assimilating C(1)-C(5) fatty acids, alcohols, and intermediates of the tricarboxylic acid cycle. Strain AZ1 could also grow photoorganotrophically with acetate as the sole photosynthetic electron donor. Chemotrophic growth in the dark under microoxic conditions was not detected. Optimum growth occurred at pH 6.5-6.7, 30-35 degrees C, > or =50 micro mol quanta m(-2) s(-1), and 2.4-2.6% NaCl. The DNA base composition was 64.5 mol% G+C. Comparative sequence analysis of the 16S rRNA gene confirmed that the isolate is a member of the family Chromatiaceae. Sequence similarity to the most closely related species, Thiorhodococcus minor DSMZ 11518(T), was 97.8%; however, the value for DNA-DNA hybridization between both strains was only 20%. Because of the low genetic similarity and since strain AZ1 physiologically differs considerably from all other members of the Chromatiaceae, including Trc. minor, the new isolate is described as a new species of the genus Thiorhodococcus, Thiorhodococcus drewsii sp. nov.     

Oxysterol-induced toxicity in R28 and ARPE-19 cells

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0–50 µg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe–based assay (2,7-dichlorodihydrofluorescein diacetate [H₂DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 µg/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol–treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.     

Comparative pathogenesis of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus in noctuid hosts of different susceptibility

Neonate larvae of the noctuid moth Spodoptera exigua were susceptible to an infection by Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). Biological activity (LD(50),ST(50)) of the virus was considerably reduced as compared to its activity in the homologous host, H. armigera. Pathogenesis was studied using a recombinant HaSNPV carrying a green fluorescent protein gene, which induces fluorescence in infected cells to mark infection. In larvae of H. armigera, fluorescence was pronounced in the fat body after 2.9 days post infection and could also be detected in several other tissues. In contrast, fluorescence was not observed in tissues of S. exigua until 9 days post infection and was restricted almost exclusively to cells of the ganglia. Examination of serial sections of wildtype HaSNPV-infected S. exigua-larvae revealed a similar pattern of tissue tropism. Apparently, HaSNPV does not undergo the usual steps in host invasion and infection in this insect species, but targets specifically to nervous tissue.