Cloning of a chub metallothionein cDNA and development of competitive RT-PCR of chub metallothionein mRNA as a potential biomarker of heavy metal exposure

Metallothionein has been assayed in a range of aquatic animal tissues as an indicator of metal exposure. We sequenced chub (Leuciscus cephalus) metallothionein cDNA which showed over 90% homology to common carp, goldfish and stone loach and 77% homology to rainbow trout sequences for metallothionein. We then used the extended primer method to develop an accurate quantitative competitive RT-PCR assay for metallothionein mRNA. RT-PCR was used to measure metallothionein mRNA in feral chub from a range of field sites, with different levels of heavy metal pollution, in the West Midlands, UK. Measurements were complemented by analysis of liver and gill metallothionein protein by capillary electrophoresis. There was no significant difference in the metallothionein protein levels between fish of different rivers and there was no evidence of elevation of mRNA at the sites of highest metal exposure. The level of metal exposure (e.g. zinc, nickel and cadmium each ranging between 15 and 28 microg/l ) at the pH (7.5-8.5) of these rivers appears insufficient to elevate hepatic or gill metallothionein in chub. A lack of elevation of hepatic metallothionein mRNA in chub exposed to zinc, copper and manganese for 24 h and 10 days in the laboratory also suggests a non-responsiveness of this species.     

The profile and polarization of the coronal Lα line

We calculate the profile and polarization of the Lα line in the solar corona. Coronal temperature variation, solar wind and other non-thermal motions have been taken into account. Because of the relatively low atomic weight of hydrogen the profile of the Lα line is a sensitive indicator of the coronal temperature. The line polarization contains relatively little information except for strong magnetic fields (> 70 G).     

The NOAA Goes-12 Solar X-Ray Imager (SXI) 2. Performance

The Solar X-ray Imager (SXI) was launched on 23 July 2001 on NOAAs GOES-12 satellite and completed post-launch testing on 20 December 2001. It was brought into operations on 21 January 2003. This paper documents SXI performance and calibrations prior to an instrument degradation that occurred on 5 November 2003 and thus covers more than 420000 soft X-ray images of the Sun. This paper details component-level as well as full-system calibrations characterizing the spatial and spectral performance of the instrument, including the grazing-incidence mirror, filters, and the properties of the MCP-intensified CCD detector system. Routine image corrections are also described. These include background (dark current) subtraction, flat-fielding, off-band light-leak correction, and image pointing and timing considerations. In addition, a signal-to-noise analysis is presented. The information contained in this study is intended to enable researchers to conduct quantitative analysis of GOES-12 SXI images.     

Seasonal variation in mussel Mytilus edulis digestive gland cytochrome P4501A- and 2E-immunoidentified protein levels and DNA strand breaks (Comet assay).

Mytilus edulis digestive gland microsomes were prepared from indigenous populations sampled from a clean reference site (Port Quin) and an urban-industrial contaminated site (Blackpool) in the UK. Samples were collected in March/April, May, August and December 1998. Western blot analysis was performed using polyclonal antibodies to fish CYP1A and rat CYP2E using partially purified M. edulis CYP as a positive control, to aid identification. CYP1A- and CYP2E-immunopositive protein levels showed different site-specific seasonal variation with higher levels of CYP2E determined in May (P < 0.05). At both sites, lower levels of CYP1A-immunopositive protein but not CYP2E-immunopositive protein were observed in the samples collected in December (P < 0.05). This correlated with lower levels of nuclear DNA damage (Comet assay expressed as per cent tail DNA) observed in December compared to August (P < 0.05).     

Analysis of mutations in the p53 tumor suppressor gene and Ki- and Ha-ras proto-oncogenes in hepatic tumors of European flounder (Platichthys flesus).

DNA from five tumors, three other-injured livers and two normal liver tissue samples from the European flounder were analyzed for mutations in exons 5-8 of the tumor suppressor gene p53 and at codons 12, 13 and 61 of Ha- and Ki-ras proto-oncogenes. No tumor-specific mutations were identified by direct sequencing and single-strand conformation polymorphism of these genes. A number of silent polymorphisms were noted in p53. In addition to a need for more extensive analyses of flounder liver tumor samples for ras or p53 mutations, other cancer-related genes should be investigated.     

Elevation of cytochrome P450-immunopositive protein and DNA damage in mussels (Mytilus edulis) transplanted to a contaminated site

Mytilus edulis were collected from a reference site (Port Quin) and an urban/industrial contaminated site (New Brighton) in the UK during June 1999. Levels of PCBs (sigma7 congeners) and CB-138 were determined to be, respectively, 21 fold and 16 fold higher in the mussel digestive glands from New Brighton. Levels of CYPIA-immunopositive protein were 1.5 fold higher (P < 0.05) at the polluted site but the levels of DNA strand breaks were 1.3 fold higher (P<0.05) at the reference site. Mussels from Port Quin were placed in cages at both sites and both transplanted and indigenous populations sampled in September (13 weeks). Mussels transplanted from the reference site to the industrial site, reported elevated levels of CYP1A-immunopositive protein (1.4 fold; P < 0.05) and higher levels of DNA damage (1.2 fold; P < 0.05) compared to caged populations at the reference site and a PCB loading similar to the populations from the polluted site. Moreover, transplanted mussels had DNA damage 1.8 fold greater (P < 0.05) than indigenous mussels at the transplant site. These changes were small but significant when compared to the observed temporal changes in the indigenous populations.     

CYP1A- and CYP3A-immunopositive protein levels in digestive gland of the mussel Mytilus galloprovincialis from the Mediterranean Sea

CYP1A-immunopositive protein can be elevated in response to planar PAHs and PCBs in Mytilus sp. digestive gland whilst CYP3A-immunopositive protein has been associated with testosterone 6beta-hydroxylation in fish. Levels of CYP1A- and CYP3A-immunopositive protein were determined in Mytilus galloprovincialis digestive gland microsomes collected from 12 sites in the Mediterranean Sea during May and September 2001. CYP1A-immunopositive protein was significantly highest at contaminated sites whilst CYP3A-immunopositive protein was significantly lowest. A weak negative correlation (r2 = 0.21) was seen between CYP1A- and CYP3A-immunopositive protein. Little evidence of differences at the different sampling times was observed. These results confirm previous work indicating elevation of CYP1A-immunopositive protein in Mytilus sp. digestive gland at contaminated sites. Further study is required to characterise CYP3A-like expression in Mytilus and to elucidate the consequences of possible CYP3A-like down-regulation at contaminated sites.     

An interlaboratory comparison of piston-cylinder pressure calibration using the albite-breakdown reaction

The pressure of the reaction albite=jadeite and quartz was measured at 600° C by workers in six geophysical laboratories for the purpose of comparing pressure calibration procedures for the solid-pressure piston-cylinder apparatus. All groups used the same starting mix of crystalline reactant and products and all obtained hydrothermal reversals of the equilibrium. Solid pressure media used included talc, NaCl, boron nitride, pyrophyllite, pyrex glass and crushable ceramic. Various means of calibration were used, including internal standardization by transitions in indicator substances and the piston-in, piston-out bracketing method.There was agreement among all groups—the average preferred value of 16.3 kilobars at 600° C is enclosed by all of the error brackets assigned by the various investigators. This average preferred value is lower by nearly two kilobars than the often-quoted extrapolation of Birch and LeComte's data (1960). It will be important for both petrology and high-pressure technology to test this result in a very high gas pressure apparatus.     

Structure and physics of solar faculae

Si iv, C iv, and O vi resonance lines have been measured above quiet and active solar regions from both pointed OSO-8 instruments. From calibrated profiles, optical depths are computed with three different methods. All three methods provide evidence that the opacity above faculae is lower than above the quiet Sun. From lower and upper limits of the opacity, we derive limits of the electron density. Our first method assumes only that the source function is constant without any geometrical constraint. We find higher densities above faculae than above quiet regions (about a factor 10). A second method allows us to compute the density, temperature gradient and thickness of a plane-parallel model, for active and quiet Sun. Electron densities agree with those of the first method but they lie in the lower range of values previously determined from Skylab. This result can be explained by the moderate level activity of the observed faculae. Appendices give relevant elements of transfer theory and newly computed values of collisional rates.