Bioscience Horizons - recent issues

Editorial & Author biographies

2008-05-29

A study into the potential role of Survivin localization in resistance to drug-induced apoptosis

Angell, H. — 2008-05-29

The aim of this study was to test the hypothesis that diverting the cytoplasmic subcellular localization of the anti-apoptotic form of Survivin to the nucleus would sensitize cancer cells to chemotherapeutics. Apoptosis is a morphologically and biochemically distinctive form of cell death, critical in the maintenance of tissue homeostasis. Caspases are a family of cysteine proteases that have a vital role in the implementation of apoptosis, and their activity is regulated by Inhibitors of Apoptosis Proteins. Recent studies indicate that one such inhibitor, Survivin, may have dual functions that are specific to its cellular location, including suppression of apoptotis (cytosolic) and regulation of cell division (nuclear). Since both apoptosis and proliferation are altered in cancer, identifying whether these roles for Survivin are dependent on its subcellular localization will inform future approaches to treat chemotherapeutically resistant tumour cells. After initially confirming the specificity of several Survivin antibodies, the distribution of Survivin was examined by immunofluorescence microscopy and sub-cellular fractionation in breast cancer cell lines. The threshold of drug-induced apoptosis was compared in cells over-expressing either wildtype Survivin or a form of Survivin unable to exit the nucleus due to a mutation in its nuclear export sequence. Endogenous Survivin localized to both nucleus and cytoplasm of breast cancer cell lines. Over-expressed Survivin had an anti-apoptotic, protective function. In contrast, cells expressing Survivin with the mutated nuclear export sequence had a lower apoptotic threshold to chemotherapeutic drugs. These results demonstrate for the first time that Survivin is localized to both the nucleus and cytoplasm of breast cancer cell lines. Importantly, the sensitivity of cells to chemotherapeutic drugs was increased when Survivin's localization was restricted to the nucleus, consistent with cytoplasmic Survivin having the anti-apoptotic role. Since clinical studies have shown that nuclear Survivin is a positive prognostic factor in breast cancer patients, the data suggest that strategies to alter Survivin distribution may be useful in the fight against cancer.

Sphingosine-1-phosphate activation of TRPC5 in vascular smooth muscle cells

Fahy, S. — 2008-05-29

Calcium signalling is a complex and diverse system utilized in many cellular processes and in the transmission of cellular information. A number of transient receptor potential (TRP) proteins have been identified in humans and other mammals; these proteins are implicated as having a role in calcium signalling. TRPC5 is a member of this protein family which combines with TRPC1 to form non-selective cation channels in human saphenous vein cells, a type of smooth muscle cell. The exact function of TRPC5 remains elusive, however, it can be activated by sphingosine-1-phosphate (S1P), an endogenous signalling phospholipid involved in SMC migration.

The aim of these experiments was to investigate the effects of S1P on the intracellular calcium concentration, [Ca²⁺]_i, in HSV cells, utilizing dominant-negative (DN)-TRPC5 transfected cells to establish the role played by TRPC5 in this response. A secondary aim was to establish the effect of SMC migration on the above response parameters to S1P using the HSV scratch assay, where a ~2 mm line of cells was scraped away from the surface of a glass cover slip and the remaining cells incubated for 24 h.

Concurrent with the literature, S1P evoked a significant response in HSV cells (n=23; P=0.001). The baseline was significantly lower in the DN-TRPC5 cells compared with the control cells (P<0.001), and the maximum response in the DN-TRPC5 transfected HSV cells reached only 60% of the maximum response in control cells. This suggested that TRPC5 was involved with maintaining basal [Ca2+]_i levels and indicated the proportion of the response for which TRPC5 was responsible.

The response to S1P was significantly larger in migrated (n=7) compared with static (n=11) HSV cells (P=<0.001) and this response was delayed by ~2.3 min; the baseline was also higher in the latter group. This suggested a functional change in the cell following migration that may have been attributable to TRPC5, for example, channel up-regulation.

In conclusion, TRPC5-like channels are responsible for a proportion of the S1P response and are implicated in SMC migration. This highlights potential pharmacological targets for the treatment of atherosclerosis, neointimal hyperplasia and coronary heart disease.

The potential effect of excessive coffee consumption on nicotine metabolism: CYP2A6 inhibition by caffeic acid and quercetin

Woodward, G. M. — 2008-05-29

Over the past decade interest in the biochemical and biological properties of polyphenols has grown considerably, as epidemiological evidence for their beneficial effects on health continues to increase. Dietary polyphenol antioxidants are reported to have many ‘health promoting’ properties, including anti-inflammatory, vasoprotection, anti-cancer and anti-obesity effects. However, their absorption and metabolism are as yet not fully elucidated, particularly with regard to their interactions with other metabolized compounds, such as nicotine. Epidemiological studies have shown that coffee-drinking cigarette smokers are more likely to smoke less in comparison to non-coffee drinking cigarette smokers. Nicotine is primarily metabolized by the hepatic cytochrome P450 enzyme CYP2A6 and may be inhibited by the metal chelating properties of polyphenols via haem interactions with their hydroxyl groups. To model nicotine metabolism, bovine liver microsomes, shown to suitably represent human CYP2A6 metabolism, were isolated by calcium precipitation and differential centrifugation. Fluorometric analysis of 7-hydroxycoumarin, the CYP2A6 metabolite of coumarin and known probe for CYP2A6 activity, was used to model nicotine metabolism in vitro and to quantify the degree of CYP2A6 inhibition imposed by caffeic acid and quercetin. It was found that both caffeic acid and quercetin, major polyphenolic constituents of caffeine containing beverages, significantly inhibited CYP2A6 activity in vitro by 37.9% (P < 0.05) and 48.2% (P < 0.05), respectively. Thus, this study demonstrates the first reported biochemical evidence in support of previous epidemiological observations, where it is suggested that polyphenol interaction with CYP2A6 prolongs the pharmacological effects of nicotine by decreasing its rate of elimination.

Temperature stress-induced bleaching of the coralline alga Corallina officinalis: a role for the enzyme bromoperoxidase

Latham, H. — 2008-05-29

Coralline algae are important components of coral reefs and are involved in reef building via calcification, cementation, the synthesis of anti-fouling compounds and the synthesis of allochemicals to aid recruitment, settling and metamorphosis of reefs species. Using Corallina officinalis we have shown that these algae undergo temperature-related bleaching at similar temperatures to those known to cause bleaching in corals. The bleaching appears to be associated with considerable increases in the vanadium-containing enzyme bromoperoxidase (VBPO). This enzyme is involved in hydrogen peroxide (H₂O₂) elimination and generates the powerful brominating/oxidizing agent hypobromous acid (HOBr, probably present as Br⁺). This is used to synthesize volatile halogenated organic compounds (VHOCs) from a pool of organic acceptor molecules. Earlier in vitro work has shown this enzyme to be effective in bleaching the phycobilin photosynthetic accessory pigments and to be partly located in chloroplasts. The data presented here supports the suggestion that increases in temperature lead to an increase in the cellular production of H₂O₂ and other reactive oxygen species that result in an increase in VBPO, a subsequent increase in HOBr/Br⁺ followed by pigment bleaching when the capacity to produce VHOCs has been exceeded. Addition of the exogenous antioxidant mannitol decreases both pigment bleaching and VBPO induction. A scheme is presented to illustrate proposals for the involvement of VBPO in the bleaching of coralline algae such as C. officinalis. The importance of these species in reef building and rebuilding is discussed.

Effects of modulators of TASK potassium channels on rat pulmonary artery tone

Shah, S. D. — 2008-05-29

TWIK-related acid-sensitive potassium (TASK) channels have been implicated as having a role in maintaining and mediating the tone of pulmonary arteries by influencing the membrane potential of the smooth muscle cells. Inhibition of these channels would be expected to promote depolarization, calcium influx and contraction. The purpose of this study was to investigate the effects of TASK modulators on rat intrapulmonary artery tone. The modulators included pH, an important physiological TASK modulator, and drugs that inhibit TASK channels, such as bupivacaine, methanandamide and zinc. Small vessel myography was used to measure the tone of both conduit and resistance pulmonary arteries. Cumulative bupivacaine and methanandamide dose–response curves were compared with phenylephrine (a sympathomimetic vasoconstrictor). The effects of pH were investigated on vessel tone and responses to bupivacaine, methanandamide or zinc chloride. Bupivacaine and methanandamide (>10 µM) resulted in increased artery tone, with similar effects seen in conduit and resistance vessels. Zinc had no effect, possibly reflecting an inhibitory action on calcium channels. In the presence of endothelial blockers, methanandamide (100 µM) still resulted in an increase in artery tone, implying an action on smooth muscle. The application of nifedipine resulted in the inhibition of the response seen with bupivacaine (100 µM), implying that voltage-gated calcium entry was involved. Changing the pH from 7.3 to 8.3 resulted in vasoconstriction, and a relaxation was seen in acidic conditions. This is opposite to the result expected for TASK channel modulation, but may reflect the multiple effects of pH on smooth muscle. The contractions seen with bupivacaine and methanandamide were increased at pH 8.3 but inhibited at pH 6.3, consistent with an effect on TASK channels. Responses to bupivacaine and methanandamide were, however, very small, suggesting that currents produced by TASK channels may not be a major factor in contracting intrapulmonary arteries, but may still have a minor role.

The effects of immune challenge on phenoloxidase activity in locust salivary glands in vitro

Wells, K. L. — 2008-05-29

Homogenates of salivary glands from Locusta migratoria possess phenoloxidase (PO) activity. This study investigates the activation of prophenoloxidase (PPO) in these glands in vitro. When freshly dissected salivary glands from L. migratoria are incubated with the immunogen laminarin, and then homogenized, a ~4-fold increase in PO activity (expressed as a percentage of the total PO) can be measured within 20 min. Addition of laminarin to the incubation medium is best made prior to addition of salivary gland tissue, because when laminarin is added 10 min after the addition of tissue, the response to laminarin is reduced by ~50%. When salivary glands are incubated in Ca²⁺-free Ringer, the response to laminarin cannot be demonstrated. Addition of a calcium ionophore to the incubation in normal Ringer does not initiate a response on its own, but does augment the response to laminarin. Addition of phorbol ester to an incubation containing normal Ringer has no effect on PO activity, and does not augment the response to laminarin. In contrast, addition of okadaic acid to normal Ringer has no effect on its own, but does augment the response to laminarin. Activation of PPO in response to laminarin is therefore calcium-dependant, possibly involving an influx of extracellular Ca²⁺, and modulated by protein phosphatases. Future work should aim to clarify the function of salivary glands in the immune defence of the locust and to investigate the exact source of the PO associated with the glands.

Species richness and aggregation effects on the productivity of ruderal plant communities under drought perturbation

Nakamura, N. — 2008-05-29

The effect of species richness and spatial aggregation on the stability of community productivity in response to drought perturbation was investigated with experimental plant communities. Communities comprising all single- and three-species combinations of the ruderal species, Capsella bursa-pastoris, Tripleurospermum inodorum, Poa annua, and Stellaria media, were established in glasshouse. Habitat patchiness was manipulated by applying different seed-sowing patterns, either aggregated or random. After the establishment of communities, 8 days of drought treatment was imposed. Followed by a week of recovery with a regular watering regime, aboveground biomass was harvested. Community biomass was not affected by species richness or by aggregation, but was affected by perturbation. When multi-species community productivity was compared with monocultures in relative terms, species mixtures performed better in drought-induced conditions. This suggests that the positive effect of species richness may be enhanced under the perturbed condition. Sampling effects were evident under perturbation favouring the least productive species, P. annua and drought-tolerant S. media. All species except C. bursa-pastoris showed reduced productivity in species mixtures, but this may be mitigated under perturbed environments by species complementarity. Lack of clear responses to aggregation may suggest that the revealed diversity effect is not related to spatial structure. While competition predominates in communities in the resource-rich environment, drought perturbation enhance overall community productivity via a shift in relative significance of species interactions from competition to sampling and complementarity effects.

Body surface temperature and length in relation to the thermal biology of lizards

Garrick, D. — 2008-05-29

This study investigated body surface temperature (T_bs) in 22 lizards of 18 species. The difference between T_bs and ambient temperature (T_a) was correlated with size. The greater T_bs – T_a differentials, which were recorded in larger lizards, may occur as a result of heat transfer from the core to surface in prevention of overheating. The structure of the integument may contribute to heat dissipation. Heliothermy and thigmothermy as forms of thermoregulation were also incorporated into the data set. Heliothermic lizards showed a positive correlation between length and T_bs – T_a. Thigmothermic lizards, however, exhibited a negative correlation. Differences in size and rate of conductive heat transfer are put forward as possible reasons for the negative correlation.

The importance of birds in the diet of otter Lutra lutra on Shapwick Heath

de la Hey, D. C. — 2008-05-29

This study investigates the importance of avian species in the diet of Shapwick Heath's otter population. The frequency occurrence of prey items was recorded from spraints collected between July 2006 and January 2007. Bird feathers extracted from otter spraints were investigated under a microscope. Where possible, feathers were identified to species level. The presence of fish scales in spraints was recorded, but not quantified. Bird feathers were recorded in 41% of all spraints, compared with 4.6% on Slapton Ley in 1981 and 4.7% on the Somerset Levels in 1975. The highest proportion of bird feathers was recorded in July 2006, at 61%. Ralliformes were more frequently recorded than any other family of birds. Coot Fulica atra L. were more frequently recorded then any other species; 15 spraints were found to contain coot feathers. Mallard Anas platyrhynchos feathers were recorded in six spraints. Cormorant Phalacrocorax carbo feathers were found in three spraints and Water rail Rallus aquaticus feathers were found in two spraints. Pintail Anas acuta, teal Anas creca, moorhen Gallinula chloropus and little grebe Tachybaptus ruficollis feathers were found in one spraint each. Two spraints contained feathers from both mallard and coot, respectively. The high proportion of birds in the otter's diet, in comparison with past studies suggests that otters are substituting birds for part of their traditional diet of fish. It is recommended that studies into the nature of the fish stock and the development of the aquatic ecosystem on Shapwick Heath be undertaken.

The effect and interaction of enhanced nitrogen deposition and reduced light on the growth of woodland ground flora

Andrews, N. R. — 2008-05-29

Woodland ground flora species Geranium robertianum, Prunella vulgaris and Deschampsia caespitosa were grown in greenhouse conditions under various light regimes (100%, 20% and 5% of ambient summer light) and were misted weekly with solutions of ammonium nitrate to simulate increased atmospheric wet and dry deposition of nitrogen (N) due to pollution at 0, 15 and 30 kg ha^–1 year^–1 concentrations. In the last 5 weeks of the study, the photosynthetic rate of G. robertianum was measured weekly. At the end of the 10-week-treatment period, the plants were harvested and growth was recorded using various measurable growth parameters. Growth of the plants and photosynthetic rate of G. robertianum were found to be primarily dependent on light availability, where reduced light levels showed a decrease in overall plant growth, indicating the importance of light in woodlands. N was not found to have an effect on the growth of the plants and thus questions arise over the calculations of critical loads of N deposition for woodland habitats. Five interactions were noted where increased N led to a significant reduction in growth at 20% light, yet a significant increase in growth at 100% light. These interactions were mainly noted for P. vulgaris, which could be explained by relatively large differences between its Ellenberg values for light and N, reflecting a preference for high light conditions and fairly infertile soils. G. robertianum was least affected by a reduction in light, reflecting its preference for semi-shaded conditions.

Has golf-course management had an effect on the plant species composition and character of a calcareous grassland?

Worster, M. P. — 2008-05-29

Lowland calcareous grassland in the UK is a priority habitat designated under the UK Biodiversity Action Plan. Calcareous grasslands used to be common in north-western Europe and were utilized for grazing livestock such as sheep and cows. Many calcareous grasslands in England have been either agriculturally ‘improved’ through the addition of fertilizer and herbicides to increase productivity or have been ploughed up to make way for arable production, which has led to a dramatic decrease in the area of calcareous grassland.

Minchinhampton Common is an area of lowland calcareous grassland located within the English county of Gloucestershire. In 1889, a golf course was laid out on the common for the use and enjoyment of local people.

The first objective of this project was to investigate whether the presence of the golf course on Minchinhampton Common and associated golf-course management activities have had an effect on the composition and character of the calcareous grassland. The second objective was to investigate whether abandoned golf-course features on Minchinhampton Common, such as old fairways and old greens can recover to resemble undisturbed calcareous grassland.

Significant differences were found between the undisturbed calcareous grassland and the fairways, greens and pathways in terms of vegetation height, plant species composition, botanical diversity and soil characteristics. This demonstrates that the presence of the golf course and the management carried out to maintain it has had an effect on the composition and character of the calcareous grassland.

Significant differences were found when the fairways and greens were compared with the abandoned old fairways and old greens. This shows that after 15 years of abandonment the old fairways and old greens are no longer similar to the fairways and greens, but they are still different from the undisturbed calcareous grassland. It is hypothesized that to aid recovery, the abandoned features should be cut annually and the clippings removed. This will decrease the nutrient status of the soil and reduce the competitiveness of grasses, thus providing greater opportunities for the establishment of forb species typical of nutrient poor calcareous grasslands in north-western Europe.

The role of hepatitis C virus in the pathogenesis of hepatocellular carcinoma

Tran, G. — 2008-05-29

Hepatitis C virus (HCV) is a worldwide pandemic, chronically affecting over 170 million people worldwide. It is a major risk factor for the development of hepatocellular carcinoma (HCC) and there is increasing experimental evidence to suggest that the virus plays a direct role in neoplastic transformation. The purpose of this letter is to review the literature regarding two individual proteins of HCV, namely NS5A and core, and their role in the pathogenesis of HCC through perturbations of cellular pathways, in addition to their immunopathological effects of chronic inflammation. A systematic search of MEDLINE in addition to manual searches of citations in key papers was employed to identify relevant studies. There is overwhelming evidence suggesting the direct and indirect roles HCV plays in the pathogenesis of HCC. Recent progress in our understanding of the pathophysiology of HCV coupled with advances in in vitro models will ensure that positive strides are made in the treatment and management of this potentially fatal virus.

Rosiglitazone-induced SERCA2b inhibition: implications for monocyte cytoskeletal remodelling and diabetic microangiopathy

Atkin, L. — 2008-03-01

In type 2 diabetes, exposure of monocytic cells to inflammatory stimuli can lead to aberrant cytoskeletal remodelling and contribute to the development of diabetic microangiopathy. Previous studies within our research group have demonstrated that Rosiglitazone significantly reduces formyl-methionyl-leucyl-proline (f-MLP)-induced actin polymerization in monocytes by increasing cytosolic calcium ion concentrations ([Ca²⁺]_i) in both resting and f-MLP-stimulated cells. As the timescale of this effect resembled inhibition of SERCA2b, the ‘housekeeping’ Ca²⁺ pump enzyme that sequesters Ca²⁺ into the ER compensatory to non-specific leakage into the cytoplasm, the present study sought to test the hypothesis that Rosiglitazone-induced increases in [Ca²⁺]_i are brought about by inhibition of SERCA2b, and also investigated how such changes are transduced into cytoskeletal remodelling.

Coupled-enzyme Ca²⁺-ATPase assays revealed a SERCA2b activity of 49 ± 5 nmol/mg/min in monocytic microsomes but pre-incubation with Rosiglitazone (20 µm; 30 min) induced statistically significant inhibition of this activity (P < 0.05).

Permeabilization of cultured monocytes in external solutions containing differing [Ca²⁺]_free was used to manipulate [Ca²⁺]_i, and F-actin content measured using flow cytometric analysis of FITC-phalloidin fluorescence. At [Ca²⁺]_i >100 nm, a significant decrease in cells' F-actin content was observed (P < 0.05). In conditions of minimal [Ca²⁺]_i, f-MLP exerted a polymerizing effect on the actin cytoskeleton; however, as [Ca²⁺]_i was increased to >100 nm, this polymerizing effect was significantly reduced.

Finally, as a literature search identified the Ca²⁺-dependent actin-modulating protein gelsolin as a candidate for transducing changes in [Ca²⁺]_i into actin cytoskeletal remodelling, western blot experiments using anti-gelsolin antibodies were used to detect ~88 kDa immunogenic bands in monocytic protein extracts, thus confirming the expression of gelsolin in monocytes.

In conclusion, the current study demonstrates the importance of [Ca²⁺]_i in modulating actin cytoskeletal remodelling, and suggests that Rosiglitazone, via its ability to affect monocytic Ca²⁺ signalling processes, may confer a level of protection against the development of diabetic microangiopathy.

Binding of (-)-epigallocatechin-3-gallate to the Hsp70 ATPase domain may promote apoptosis in colorectal cancer

Brierley-Hobson, S. — 2008-03-01

Colorectal cancer (CRC) patients frequently have a poor prognosis because of metastases and drug resistance. Heat-shock protein 70 (Hsp70) over-expression in cancer may exacerbate these factors by inhibiting apoptosis, thus providing a potential therapeutic target. The green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG) induces apoptosis in numerous cancer cell lines by unclear mechanisms. This study proposed and investigated a novel mechanism by which EGCG might inhibit the anti-apoptotic activity of Hsp70, namely, by competing with adenosine triphosphate (ATP) for binding to the Hsp70 ATPase domain. It also examined the impact of EGCG on the CRC cell line HT-29, which is known to over-express Hsp70.

HT-29 cells were treated with 10–300 µm EGCG for 8, 24 and/or 48 h, then analysed using an MTS proliferation-assay or flow cytometry with Annexin V-FITC and propidium iodide. The competitive binding of EGCG to the Hsp70 ATPase domain was assessed using ATP-agarose, a dot blot and chemiluminescence techniques.

EGCG significantly (P < 0.001) and dose-dependently inhibited HT-29 cell viability. Viability was inhibited by 50% (IC₅₀) at 89 µm and 74 µm after 24- and 48-h treatments, respectively, also suggesting a time-dependent effect. Apoptosis was induced both dose- and time-dependently, commencing at 50 µm after 8- and 24-h treatments. Further, apoptosis correlated significantly (P < 0.01) with reduced viability as measured by MTS at 24 h, indicating 98% causality. The dot blot suggested 200 µm EGCG competed with ATP for binding to Hsp70, presumably by binding its ATPase domain, theoretically implying this flavonoid could inhibit the anti-apoptotic effect of Hsp70.

Morphological and immunophenotypic analysis of basal-like carcinoma of the breast

Winter, J. — 2008-03-01

Breast carcinomas present a heterogeneous group of tumours varying greatly with regard to histopathological classification, prognosis and treatment options. Recent research has identified a subtype of breast carcinoma that is associated with a poor prognosis and is most commonly found to be grade 3; these tumours are termed basal-like tumours due to their differentiation towards basal cell types.

Sixty cases of grade 3 ductal carcinomas not otherwise specified (NOS) were stained using immunohistochemical techniques with a panel of antibodies.

All cases were subjected to immunohistochemical and morphological analyses, and tumours were classified as either basal-like or ductal NOS.

Statistical analysis accompanied by sensitivity and specificity values showed cytokeratin 5 and vimentin to be the most useful antibodies for diagnosing the basal-like tumour. Morphological analysis found that a high mitotic count and presence of necrosis could be associated with the basal-like phenotype.

Results of this study have facilitated the development of an algorithm to allow identification and diagnosis of this tumour type.

Gene expression of Pseudomonas aeruginosa and MRSA within a catheter-associated urinary tract infection biofilm model

Goldsworthy, M. J. H. — 2008-03-01

Catheter-associated urinary tract infections (CAUTI) have frequently been studied in monocultures in vitro. However, many CAUTI are due to the presence of a mixed microbial community and not just a single population, especially within patients subjected to long-term catheterization. This can potentially have important clinical implications in regards to treatment strategy and outcome. This study revealed that when Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) (both commonly found in CAUTI) were grown together as a mixed culture within a CAUTI model in comparison to their respective monocultures, accelerated biofilm development was observed. Virulence gene expression analysis within P. aeruginosa and MRSA monoculture and mixed biofilms was performed through use of real-time quantitative PCR. It was revealed that production of P. aeruginosa exotoxin A was increased 1839-fold when P. aeruginosa and MRSA were grown together as a mixed biofilm. Significant expression of -haemolysin by MRSA was not observed in either culture. It is proposed that both the biofilm-forming capability and virulence gene expression of microbes within CAUTI mixed biofilms may differ substantially to the respective microbial monocultures. This highlights the importance of developing specific treatment strategies for CAUTI when polymicrobial communities are present.

Dissolution kinetics of clinoptilolite and heulandite in alkaline conditions

Cotton, A. — 2008-03-01

Temperature and pH-dependent dissolution kinetics of natural clinoptilolite and a ‘pure’ heulandite phase have been determined and compared. Si dissolution rates increased with increasing temperature and pH. Heulandite has a slightly higher dissolution rate than clinoptilolite in the pH range 8–10. Measured reaction order was 0.23 and 0.14 for clinoptilolite and heulandite, respectively. This is slightly lower than for other aluminosilicate phases at comparable temperatures. The measured dissolution rates indicate a 1 mm spherical crystal would dissolve in 38 848 years and 17 301 years for clinoptilolite and heulandite, respectively. If solution composition is maintained, and remains unsaturated with respect to Si, Al, etc., the presence of zeolites in the vicinity of an underground nuclear waste repository would be beneficial as a sorption barrier.

Factors controlling internal initiation of transcription at PRY3 in budding yeast

Lee, B. — 2008-03-01

Transcription initiation from within the coding regions of genes has been observed in budding yeast as a natural phenomenon induced by environmental changes. However, such ‘internal initiation’ events have also been observed in mutant yeast strains with aberrant chromatin structure. Failure to suppress initiation from cryptic internal promoters is thought to result from either loss of appropriate nucleosome density or loss of appropriate histone modifications in the coding region. PRY3 is a gene previously shown to undergo internal initiation in response to mating pheromone. This project aimed to uncover which factors are involved in suppressing inappropriate transcription initiation from an internal TATA-box at PRY3 in the absence of mating pheromone. This work demonstrates that factors controlling nucleosome density (Spt6, Spn1 and Spt10) but not histone deacetylation (Eaf3) are required to suppress internal initiation at PRY3 in the absence of mating pheromone. In addition, TATA-box binding protein (TBP) is required for internal initiation at PRY3. Taken together, these results indicate that internal initiation at PRY3 is predominantly suppressed by a nucleosome reassembly mechanism rather than histone modifications, and suggest that the transcriptional output at PRY3 is controlled by competition between TBP and nucleosomes for binding an internal TATA-box.

The Klebsiella pheV tRNA locus: a hotspot for integration of alien genomic islands

van Aartsen, J. J. — 2008-03-01

Klebsiella sp. cause a wide range of human infections, particularly nosocomial septicaemia, pneumonia and urinary tract infections. Like other Enterobacteriaceae, Klebsiella are likely to possess plastic genomes comprised of core regions interspersed with horizontally acquired genomic islands. As phenylalanine tRNA genes are known to be occupied by islands in other Enterobacteriaceae, we utilized PCR-based screening and chromosome walking techniques to examine the pheV locus in Klebsiella isolates from blood stream and urinary tract infections. We hypothesized that this gene was an integration hotspot that served as a repository for novel genetic material in Klebsiella. The pheV site in Klebsiella KR116 and KR164 harboured an islet encoding four genes, two with similarity to genes within an island downstream of pheR in Salmonella enterica serovar Typhi CT18. In KR173 the locus contained a larger, potentially intact version of this island and harboured an integrase gene similar to that in the S. Typhi CT18 island. However, the Klebsiella and Salmonella islands were clearly distinguishable by strain-specific segments and organizational variation. On the basis of available sequence and restriction fragment length polymorphism data, three other Klebsiella isolates were found to possess an entirely distinct entity that resembled a 12.6 kb pheV associated island in K. pneumoniae MGH78578. This island was predicted to encode a P pilus-like structure, a probable virulence factor on the basis of parallels with E. coli. A unique and intriguing feature of Klebsiella pheV loci was the presence of multiple tandem repeats of up to 163 bp immediately downstream of pheV and a truncated copy at the opposite end of the islands. The tRNA proximal repeats were variable in number and size between isolates, while the solitary downstream repeats varied in length. These elements may represent genetic debris of previous recombination events. In conclusion, the pheV locus of Klebsiella exhibited considerable variability between strains and harboured at least two distinct island types that could play important roles in adaptation and/or virulence. Functional characterization of this genetic armory will help unravel basic microbial and pathogenesis processes and may in time lead to improvements in the diagnosis, prevention and treatment of Klebsiella infections.

An investigation to determine variation in marsh orchid (Dactylorhiza) populations at Moses Gate Country Park, Bolton

Crompton, C. — 2008-03-01

In June 2006, two populations of Dactylorhiza were examined at Moses Gate Country Park, in Greater Manchester, to determine whether variation exists between the two populations. Careful measurements of orchid characteristics were taken at both sites, in addition to soil pH recordings, and the results compared and tested for significance using one-way ANOVA and Chi-square significance tests.

Descriptive statistics and box-plots showed that there were indeed differences in orchid height, leaf width and leaf number between the two populations, with one-way ANOVA confirming their significance as P < 0.001 for all the variables. Further analyses using frequency data and Chi-square tests also showed that there were significant variations in spur size, leaf type, labellum shape and petal colour between the two sites with P < 0.001 for all these characteristics. Soil pH data were also analysed and tested using one-way ANOVA which showed that there were significant variations in soil pH levels at both sites with P < 0.001 for this data.

It was concluded that there were indeed significant differences between the two populations of Dactylorhiza at Moses Gate Country Park, therefore the null hypothesis was rejected. It was suggested that further investigations could be undertaken at this site, using similar methods, over a number of years, to support this study and to provide further constructive information on the status of the two populations of orchids, which may increase the conservation value of the site.

Is the clinical use of adult stem cells a realistic possibility for myocardial regeneration?

Kaczmarczyk, K. — 2008-03-01

Cardiac disease is the leading cause of death in the Western World and as such presents a large incentive for finding a cure. There is much current interest in exploiting adult stem cells in cardiac therapies, with both heart tissue and bone marrow being considered as potential sources or stem cells. Some promising results for future therapies are emerging, but it remains unclear how long it will take for the research to translate into clinical use. Further basic research into stem cell biology needs to be conducted before such therapies can be used with confidence.

A dynamic website for a government/industry-funded project exploring biofortification of wheat with selenium

Adams, E. J. — 2008-03-01

In 2005, scientists within several UK-based academic and industrial organizations came together to work on a research project, funded jointly by the UK Department for Environment, Food and Rural Affairs and several industry partners. The project was entitled Biofortification through Agronomy and Genotypes to Elevate the Levels of Selenium (BAGELS). The aim of the BAGELS project, which will report in 2009, is to determine if it is possible to increase safely the selenium (Se) content of bread, through the use of Se-containing fertilizers (i.e. agronomic biofortification). The BAGELS project is also seeking to study the natural genetic variation in grain Se concentration to determine if it might be possible to breed for Se-enriched wheat (i.e. genetic biofortification). The underlying rationale for the BAGELS project is that Se-biofortified wheat is a potential strategy for increasing the dietary intakes of Se and thereby improve human health for most of the population. It was recognized that an official website for the BAGELS project could be used to communicate the importance of Se to human health in addition to providing a simple means to exchange results between scientists. Therefore, the aim of my research project was to create a BAGELS project website, which delivered specific objectives to meet the needs of the project manager and project consortium members. After investigation into website design and construction, accessibility and web-authoring tools, a final dynamic website was created using the free open source software Joomla!. A review of the recent scientific studies on Se and health, and the options for increasing Se intake were included as a first step towards developing a source of public information. There is strong evidence linking low Se intake and status in humans and adverse health effects, including immune disfunction and cancer.