Rare insights into cancer biology.

Cancer-associated mutations have been identified in the metabolic genes succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH), advancing and challenging our understanding of cellular function and disease mechanisms and providing direct links between dysregulated metabolism and cancer. Some striking parallels exist in the cellular consequences of the genetic mutations within this triad of cancer syndromes, including accumulation of oncometabolites and competitive inhibition of 2-oxoglutarate-dependent dioxygenases, particularly, hypoxia-inducible factor (HIF) prolyl hydroxylases, JmjC domain-containing histone demethylases (part of the JMJD family) and the ten-eleven translocation (TET) family of 5methyl cytosine (5mC) DNA hydroxylases. These lead to activation of HIF-dependent oncogenic pathways and inhibition of histone and DNA demethylation. Mutations in FH, resulting in loss of enzyme activity, predispose affected individuals to a rare cancer, hereditary leiomyomatosis and renal cell cancer (HLRCC), characterised by benign smooth muscle cutaneous and uterine tumours (leiomyomata) and an aggressive form of collecting duct and type 2 papillary renal cancer. Interestingly, loss of FH activity results in the accumulation of high levels of fumarate that can lead to the non-enzymatic modification of cysteine residues in multiple proteins (succination) and in some cases to their disrupted function. Here we consider that the study of rare diseases such as HLRCC, combining analyses of human tumours and cell lines with in vitro and in vivo murine models has provided novel insights into cancer biology associated with dysregulated metabolism and represents a useful paradigm for cancer research.

Comparative bioenergetic assessment of transformed cells using a cell energy budget platform.

The aberrant expression and functional activity of proteins involved in ATP production pathways may cause a crisis in energy generation for cells and compromise their survival under stressful conditions such as excitation, starvation, pharmacological treatment or disease states. Under resting conditions such defects are often compensated for, and therefore masked by, alternative pathways which have significant spare capacity. Here we present a multiplexed 'cell energy budget' platform which facilitates metabolic assessment and cross-comparison of different cells and the identification of genes directly or indirectly involved in ATP production. Long-decay emitting O(2) and pH sensitive probes and time-resolved fluorometry are used to measure changes in cellular O(2) consumption, glycolytic and total extracellular acidification (ECA), along with the measurement of total ATP and protein content in multiple samples. To assess the extent of spare capacity in the main energy pathways, the cells are also analysed following double-treatment with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone and oligomycin. The four-parametric platform operating in a high throughput format has been validated with two panels of transformed cells: mouse embryonic fibroblasts (MEFs) lacking the Krebs cycle enzyme fumarate hydratase (Fh1) and HeLa cells with reduced expression of pyrimidine nucleotide carrier 1. In both cases, a marked reduction in both respiration and spare respiratory capacity was observed, accompanied by a compensatory activation of glycolysis and consequent maintenance of total ATP levels. At the same time, in Fh1-deficient MEFs the contribution of non-glycolytic pathways to the ECA did not change.

Cellular nicotinamide adenine dinucleotide (NADH) as an indicator of bacterial metabolic activity dynamics in activated sludge.

In this study, native fluorescent nicotinamide adenine dinucleotide (NADH) was used as a direct indicator of bacterial metabolic activity in activated sludge. Specific NADH concentration was dynamic and varied between 10(6)-10(8) molecules per bacterial cell. Low concentrations (10(6)-10(7) NADH molecules cell(-1)) indicate efficient bacterial metabolic activity while high concentrations (10(7)-10(8) NADH molecules cell(-1)) indicate inefficient bacterial metabolic activity. Specific [NADH] did not correlate to changes in dissolved organic carbon, but increases correlated to decreases in oxygen uptake rates. Perhaps a lack of oxygen as the terminal electron acceptor prevented efficient reoxidization of NADH to NAD+, which resulted in an accumulation of NADH within the cells. Also, significant amounts of NADH were released and accumulated into the extracellular medium of metabolically active E. coli cells in log phase. Such overflow metabolism may be the product of favourable conditions. Thus, the flux of both specific intracellular and extracellular [NADH] indicates the dynamics of bacterial metabolic activity in activated sludge.

Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations.

The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.

Genetic and functional analyses of FH mutations in multiple cutaneous and uterine leiomyomatosis, hereditary leiomyomatosis and renal cancer, and fumarate hydratase deficiency.

Germline mutations of the fumarate hydratase (FH, fumarase) gene are found in the recessive FH deficiency syndrome and in dominantly inherited susceptibility to multiple cutaneous and uterine leiomyomatosis (MCUL). We have previously reported a number of germline FH mutations from MCUL patients. In this study, we report additional FH mutations in MCUL and FH deficiency patients. Mutations can readily be found in about 75% of MCUL cases and most cases of FH deficiency. Some of the more common FH mutations are probably derived from founding individuals. Protein-truncating FH mutations are functionally null alleles. Disease-associated missense FH changes map to highly conserved residues, mostly in or around the enzyme's active site or activation site; we predict that these mutations severely compromise enzyme function. The mutation spectra in FH deficiency and MCUL are similar, although in the latter mutations tend to occur earlier in the gene and, perhaps, are more likely to result in a truncated or absent protein. We have found that not all mutation-carrier parents of FH deficiency children have a strong predisposition to leiomyomata. We have confirmed that renal carcinoma is sometimes part of MCUL, as part of the variant hereditary leiomyomatosis and renal cancer (HLRCC) syndrome, and have shown that these cancers may have either type II papillary or collecting duct morphology. We have found no association between the type or site of FH mutation and any aspect of the MCUL phenotype. Biochemical assay for reduced FH functional activity in the germline of MCUL patients can indicate carriers of FH mutations with high sensitivity and specificity, and can detect reduced FH activity in some patients without detectable FH mutations. We conclude that MCUL is probably a genetically homogeneous tumour predisposition syndrome, primarily resulting from absent or severely reduced fumarase activity, with currently unknown functional consequences for the smooth muscle or kidney cell.

Myosin V-mediated vacuole distribution and fusion in fission yeast.

The class V myosins are actin-based motors that move a variety of cellular cargoes [1]. In budding yeast, their activity includes the relocation of a portion of the vacuole from the mother cell to the bud [2, 3]. Fission yeast cells contain numerous (approximately 80) small vacuoles. When S. pombe cells are placed in water, vacuoles fuse in response to osmotic stress [4]. Fission yeast possess two type V myosin genes, myo51(+) and myo52(+) [5]. In a myo51Delta strain, vacuoles were distributed throughout the cell, and mean vacuole diameter was identical to that seen in wild-type cells. When myo51Delta and wild-type cells were placed in water, vacuoles enlarged by fusion. In myo52Delta cells, by contrast, vacuoles were smaller and mostly clustered around the nucleus, and fusion in water was largely inhibited. When cells containing GFP-Myo52 were placed in water, Myo52 was seen to redistribute from the cell poles to the surface of the fusing vacuoles. Vacuole fusion in fission yeast was inhibited by the microtubule drug thiabendazole (TBZ) but not by the actin inhibitor latrunculin B. This is the first demonstration of the involvement of a type V myosin, possibly via an interaction with microtubules, in homotypic membrane fusion.

Managing a complex river catchment: a case study on the River Almond.

The River Almond catchment in the east central belt rates amongst Scotland's most polluted rivers. This paper describes how shifting forms of economic development in the catchment since the 1860s have affected river quality. A legacy of effects from past land uses has combined with the impacts associated with current land uses to present a substantial and complex management challenge. It is argued that there have been, and still are, scientific, legislative and socio-economic constraints on the abilities of managers to respond promptly to changing patterns of impacts on the river system. This response lag has tended to increase the costs of subsequent remediation, and has consequently supported a downward pressure on the quality standards that are considered 'realistic' by river managers. Conversely, advances in science, the expansion of regulatory powers and resources, and an increase in public interest in the environment has helped to clarify the extent of the management problems in the catchment, and allowed major advances in some areas of pollution management. In an attempt to overcome some of the remaining weaknesses of river management, river managers and interest groups have started to form inter-sectoral and multidisciplinary partnerships. The success of these new groupings is highly contingent, not so much on the competence of their scientific advice, but on the priority given to water protection by the land-use decision-makers involved, and the resources they are prepared to commit to funding rehabilitation.

Biliary PAH metabolites and EROD activity in flounder (Platichthys flesus) from a contaminated estuarine environment.

The present study in the Firth of Forth, Scotland, emphasises the usefulness of biliary metabolite measurements in estuarine monitoring using the flounder (Platichthys flesus). The short time scale (a few hours) of response to polycyclic aromatic hydrocarbon (PAH) exposure, allowed clearer interpretation of trends and differences between sites. Such differences and trends in the metabolite data were not as apparent from 7-ethoxyresorufin O-deethlyation (EROD) activity measurements, which were likely to have been blurred by the movements of flounder between sites. Statistical differences were not observed in EROD activity in flounder from the Firth of Forth (ANOVA, P = 0.065). The biliary metabolites showed statistical differences between the uppermost site of Longannet and Port Edgar, in the outer Firth, for both the 1-OH pyrene and 2-OH naphthalene metabolites (ANOVA, Tukeys, P = 0.012 and 0.022 respectively). There was also a significant correlation between the concentrations of all three individual metabolites (1-OH pyrene, 2-OH naphthalene, I-OH phenanthrene) and the log of the distance downstream from the major PAH input sources of Longannet power station and Grangemouth. Biliary PAH metabolites were shown to better reflect local contamination gradients than EROD activity. This is explained by the relative response times of the two biomarkers and local movements of the flounder in the estuary.

The influence of victim gender and sexual orientation on judgments of the victim in a depicted stranger rape.

This study investigated the impact of respondent gender, victim gender, and victim sexual orientation on judgments toward the victim of a depicted stranger rape. Respondents were required to read a scenario in which victim gender and sexual orientation varied between subjects, and to complete measures of behavioral blame, responsibility, and severity of the attack. Results revealed that male respondents made more anti-victim judgments than female respondents did. Male respondents judged gay male victims more negatively than they did other victims. Female respondents' judgments were pro-victim regardless of victim gender and victim sexual orientation. Results are discussed in relation to the feminist analysis of victim blame, and blame toward male rape victims. Implications for support services, particularly of male victims, are also considered.

Synthesis and biological activities of spiroheterocyclic growth hormone secretagogues.

The synthesis and biological activities of a series of spiroheterocyclic growth hormone secretagogues are reported. Modification of the spiroindane part-structure of the prototypal secretagogue L-162,752 revealed that the spiroindane could be replaced with spirobenzodihydrothiophen derivatives to enhance not only in vitro potency but also oral activity. In this study non-aromatic D-2-amino-4-cyclohexylbutanoic analogs (8a-8d) were also identified to be active secretagogues.

Hypoxia, hypocapnia and spirometry at altitude.

1. Both hypoxia and hypocapnia can cause broncho-constriction in humans, and this could have a bearing on performance at high altitude or contribute to altitude sickness. We studied the relationship between spirometry, arterial oxygen saturation and end-tidal carbon dioxide (ETCO2) concentration in a group of healthy lowland adults during a stay at high altitude, and then evaluated the response to supplementary oxygen and administration of a beta 2 agonist. 2. We collected spirometric data from 51 members of the 1994 British Mount Everest Medical Expedition at sea level (barometric pressure 101.2-101.6 kPa) and at Mount Everest Base Camp in Nepal (altitude 5300 m, barometric pressure 53-54.7 kPa) using a pocket turbine spirometer. A total of 205 spirometric measurements were made on the 51 subjects during the first 6 days after arrival at Base Camp. Further measurements were made before and after inhalation of oxygen (n = 47) or a beta 2 agonist (n = 39). ETCO2 tensions were measured on the same day as spirometric measurements in 30 of these subjects. 3. In the first 6 days after arrival at 5300 m, lower oxygen saturations were associated with lower forced expiratory volume in 1 s (FEV1; P < 0.02) and forced vital capacity (FVC; P < 0.01), but not with peak expiratory flow (PEF). Administration of supplementary oxygen for 5 min increased oxygen saturation from a mean of 81%-94%, but there was no significant change in FEV1 or FVC, whilst PEF fell by 2.3% [P < 0.001; 95% confidence intervals (CI) -4 to -0.7%]. After salbutamol administration, there was no significant change in PEF, FEV1 or FVC in 35 non-asthmatic subjects. Mean ETCO2 at Everest Base Camp was 26 mmHg, and a low ETCO2 was weakly associated with a larger drop in FVC at altitude compared with sea level (r = 0.38, P < 0.05). There was no correlation between either ETCO2 or oxygen saturation and changes in FEV1 or PEF compared with sea-level values. 4. In this study, in normal subjects who were acclimatized to hypobaric hypoxia at an altitude of 5300 m, we found no evidence of hypoxic broncho-constriction. Individuals did not have lower PEF when they were more hypoxic, and neither PEF nor FEV1 were increased by either supplementary oxygen or salbutamol. FVC fell at altitude, and there was a greater fall in FVC for subjects with lower oxygen saturations and probably lower ETCO2.

Molecular evidence for a common leukaemic progenitor in acute mixed lymphoid and myeloid leukaemia.

De novo acute leukaemia presenting with a mixed lymphoid and myeloid leukaemic population has rarely been described. We have used the consensus Ig heavy chain primers and DNA isolated from these two distinct populations of cells in polymerase chain reactions. We demonstrated that both populations of cells exhibited Ig heavy chain gene rearrangements. Cloning and subsequent DNA sequencing of the amplified products showed a common V-D-J junctional nucleotide sequence. This work therefore provides the first evidence that the leukaemic cells in de novo acute leukaemia with a mixed lymphoid and myeloid population are derived from a common progenitor clone and may offer an explanation for the poor prognosis in these patients.

Gide in the U.S.S.R.: some observations on Comradeship.

After 1914-18, Gide emphasized the value of the Comradeship of Mankind rather than the essentially individualistic ethos to which he had been previously committed. However, while believing in the social benefits of tolerating pederasty, he still saw a person's difference from the norm as the guarantee of authenticity. Political idealism and curiosity took him to the U.S.S.R. in 1936, and on his return he criticized the inertia, ignorance, and conformism which he considered were encouraged by the Soviet state's promotion of the family unit. This essay examines how his attitude towards sexuality led him to question alleged political freedoms and to see in the Soviet oppression of minorities, including homosexuals, the denial of the revolutionary spirit.

The source of belief bias effects in syllogistic reasoning.

In studies of the belief bias effect in syllogistic reasoning, an interaction between logical validity and the believability of the conclusion has been found; in essence, logic has a larger effect on unbelievable than on believable conclusions. Two main explanations have been proposed for this finding. The selective scrutiny account claims that people focus on the conclusion and only engage in logical processing if this is found to be unbelievable; while the misinterpreted necessity account claims that subjects misunderstand what is meant by logical necessity and respond on the basis of believability when indeterminate syllogisms are presented. Experiments 1 and 2 compared the predictions of these two theories by examining whether the interaction would disappear if only determinate syllogisms were used. It did, thus providing strong support for the misinterpreted necessity explanation. However, the results are also consistent with a version of the mental models theory, and so Experiment 3 was carried out to compare these two explanations. The mental models theory received strong support, as it did also in the follow-up Experiments 4 and 5. It is concluded that people try to construct a mental model of the premises but, if there is a believable conclusion consistent with the first model they produce, then they fail to construct alternative models.

Judgements about victims and attackers in depicted rapes: a review.

This paper reviews the effects on subjects' judgements of a variety of factors that have been included in experimental depictions of rape. The focus is on attribution of responsibility or fault to the victim or attacker and related judgements, particularly regarding guilt and sanctions. Generally, females make more pro-victim judgements than do males, and people with non-traditional sex-role attitudes make more pro-victim judgements than do holders of more traditional views. Other factors covered are various victim characteristics, victim-attacker acquaintance, resistance, and victim attire and a range of behaviours prior to the attack. These are limits to generalization due to populations studied and methods used, and the observed effects of several factors are either minimal or inconsistent. However, some factors have reliable effects on judgements, which it is argued are explainable in terms of their link with traditional beliefs about women's rights and roles. Males have often been found to be more susceptible to these effects. In particular, it appears that if a female engages in any behaviour deemed to be 'incautious' that results in victimization then she may be perceived to be at fault, even though these behaviours would be 'legitimate' for males, and that prior romantic involvement with the attacker mitigates the perceived seriousness of, and may even be seen as supplying justification for, a sexual attack. The existence of these attitudes implies that rape may be tacitly condoned in many situations.

The effect of set size on the interpretation of quantifiers used in rating scales.

Two studies are reported which investigated how people interpret quantifiers of amount such as are commonly used in questionnaires and rating scales. The results indicated that the interpretation of certain quantifiers and rating scales. The results indicated that the interpretation of certain quantifiers varied depending on the context in which they occurred. Low-magnitude quantifiers (e g, 'few', 'several') seemed to signify a much greater proportion when they described small set sizes than when they described relatively large ones. This means that it will be virtually impossible to find quantifiers for use in rating scales which achieve the desirable property of interval scaling. Despite this, some quantifiers are clearly more consistent in their interpretation and more appropriate to use than others, and recommendations are made as to the best ones to use in different situations.

The effect of secoverine hydrochloride on stimulated sigmoid motility: a double-blind, placebo controlled cross-over study in irritable bowel syndrome.

The effect of oral secoverine hydrochloride on neostigmine-stimulated sigmoid motility in 12 patients with irritable bowel syndrome was studied in a double-blind placebo-controlled cross-over study. Both spontaneous and stimulated motor activity were significantly reduced by the compound in comparison with placebo. The most sensitive indices were the frequency of wave activity, maximum amplitude and motility index. Two patients reported mild dizziness after secoverine.

Microbial biomass and productivity in seagrass beds.

Different methods for measuring the rates of processes mediated by bacteria in sediments and the rates of bacterial cell production have been compared. In addition, net production of the seagrass Zostera capricorni and bacterial production have been compared and some interrelationships with the nitrogen cycle discussed. Seagrass productivity was estimated by measuring the plastochrone interval using a leaf stapling technique. The average productivity over four seasons was 1.28 +/- 0.28 g C m-2 day-1 (mean +/- standard deviation, n = 4). Bacterial productivity was measured five times throughout a year using the rate of tritiated thymidine incorporated into DNA. Average values were 33 +/- 12 mg C m-2 day-1 for sediment and 23 +/- 4 for water column (n = 5). Spatial variability between samples was greater than seasonal variation for both seagrass productivity and bacterial productivity. On one occasion, bacterial productivity was measured using the rate of 32P incorporated into phospholipid. The values were comparable to those obtained with tritiated thymidine. The rate of sulfate reduction was 10 mmol SO4(-2) m-2 day-1. The rate of methanogenesis was low, being 5.6 mg CH4 produced m-2 day-1. A comparison of C flux measured using rates of sulfate reduction and DNA synthesis indicated that anaerobic processes were predominant in these sediments. An analysis of microbial biomass and community structure, using techniques of phospholipid analysis, showed that bacteria were predominant members of the microbial biomass and that of these, strictly anaerobic bacteria were the main components. Ammonia concentration in interstitial water varied from 23 to 71 micromoles. Estimates of the amount of ammonia required by seagrass showed that the ammonia would turn over about once per day. Rapid recycling of nitrogen by bacteria and bacterial grazers is probably important.