Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

An evaluation protocol for subtype-specific breast cancer event prediction

In recent years increasing evidence appeared that breast cancer may not constitute a single disease at the molecular level, but comprises a heterogeneous set of subtypes. This suggests that instead of building a single monolithic predictor, better predictors might be constructed that solely target samples of a designated subtype, which are believed to represent more homogeneous sets of samples. An unavoidable drawback of developing subtype-specific predictors, however, is that a stratification by subtype drastically reduces the number of samples available for their construction. As numerous studies have indicated sample size to be an important factor in predictor construction, it is therefore questionable whether the potential benefit of subtyping can outweigh the drawback of a severe loss in sample size. Factors like unequal class distributions and differences in the number of samples per subtype, further complicate comparisons. We present a novel experimental protocol that facilitates a comprehensive comparison between subtype-specific predictors and predictors that do not take subtype information into account. Emphasis lies on careful control of sample size as well as class and subtype distributions. The methodology is applied to a large breast cancer compendium involving over 1500 arrays, using a state-of-the-art subtyping scheme. We show that the resulting subtype-specific predictors outperform those that do not take subtype information into account, especially when taking sample size considerations into account.     

Regulation of p53 by metal ions and by antioxidants: dithiocarbamate down-regulates p53 DNA-binding activity by increasing the intracellular level of copper.

Mutations in the p53 tumor suppressor gene frequently fall within the specific DNA-binding domain and prevent the molecule from transactivating normal targets. DNA-binding activity is regulated in vitro by metal ions and by redox conditions, but whether these factors also regulate p53 in vivo is unclear. To address this question, we have analyzed the effect of pyrrolidine dithiocarbamate (PDTC) on p53 DNA-binding activity in cell lines expressing wild-type p53. PDTC is commonly regarded as an antioxidant, but it can also bind and transport external copper ions into cells and thus exert either pro- or antioxidant effects in different situations. We report that PDTC, but not N-acetyl-L-cysteine, down-regulated the specific DNA-binding activity of p53. Loss of DNA binding correlated with disruption of the immunologically "wild-type" p53 conformation. Using different chelators to interfere with copper transport by PDTC, we found that bathocuproinedisulfonic acid (BCS), a non-cell-permeable chelator of Cu1+, prevented both copper import and p53 down-regulation. In contrast, 1,10-orthophenanthroline, a cell-permeable chelator of Cu2+, promoted the redox activity of copper and up-regulated p53 DNA-binding activity through a DNA damage-dependent pathway. We have previously reported that p53 protein binds copper in vitro in the form of Cu1+ (P. Hainaut, N. Rolley, M. Davies, and J. Milner, Oncogene 10:27-32, 1995). The data reported here indicate that intracellular levels and redox activity of copper are critical for p53 protein conformation and DNA-binding activity and suggest that copper ions may participate in the physiological control of p53 function.     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

Fasting protects against the side effects of irinotecan but preserves its anti-tumor effect in Apc15lox mutant mice

Irinotecan is a widely used topoisomerase-I-inhibitor with a very narrow therapeutic window because of its severe toxicity. In the current study we have examined the effects of fasting prior to irinotecan treatment on toxicity and anti-tumor activity. FabplCre;Apc^15lox/+ mice, which spontaneously develop intestinal tumors, of 27 weeks of age were randomized into 3-day fasted and ad libitum fed groups, followed by treatment with a flat-fixed high dose of irinotecan or vehicle. Side-effects were recorded until 11 days after the start of the experiment. Tumor size, and markers for cell-cycle activity, proliferation, angiogenesis, and senescence were measured. Fasted mice were protected against the side-effects of irinotecan treatment. Ad libitum fed mice developed visible signs of discomfort including weight loss, lower activity, ruffled coat, hunched-back posture, diarrhea, and leukopenia. Irinotecan reduced tumor size in fasted and ad libitum fed groups similarly compared to untreated controls (2.4 ± 0.67 mm and 2.4 ± 0.82 mm versus 3.0 ± 1.05 mm and 2.8 ± 1.08 mm respectively, P < 0.001). Immunohistochemical analysis showed reduced proliferation, a reduced number of vascular endothelial cells, and increased levels of senescence in tumors of both irinotecan treated groups. In conclusion, 3 days of fasting protects against the toxic side-effects of irinotecan in a clinically relevant mouse model of spontaneously developing colorectal cancer without affecting its anti-tumor activity. These results support fasting as a powerful way to improve treatment of colorectal carcinoma patients.     

Multiple field and glasshouse assessments increase the reliability of linkage mapping of the Vf source of scab resistance in apple

 Apple scab, caused by the fungus Venturia inaequalis (Cke.) Wint., is an important disease in commercial apple production. A mapping population of 155 individuals, derived from a cross between the apple varieties ‘Prima’ (resistant)בFiesta’ (susceptible), was scored for response to the disease in replicated field and glasshouse trials throughout Europe. Twenty data sets were selected and cluster analysis was used to form a consensus score for the population fitting a 1 : 1 segregation ratio of resistance:susceptibility. The progeny were scored with molecular markers. A detailed map covering 54 cM of the ‘Prima’ linkage group containing the Vf gene for scab resistance was constructed using 24 molecular markers linked to the resistance gene. One isoenzyme marker (Pgm-1), six RFLP markers and 17 RAPD markers formed a linkage group with the consensus measure of resistance to scab. Four marker bridges were established with the corresponding ‘Fiesta’ linkage group with additional markers (one isozyme, one RFLP, three RAPD and one AFLP). A low chi-square value indicated a good fit of the marker ordering, which was in close agreement with previously reported linkage positions for some of the markers and Vf. Differences were observed in the ability of different scoring methods to resolve susceptible and resistant classes. The results obtained for the consensus classification of resistance to scab for the population may suggest the presence of virulent inocula at some sites, which could overcome the Vf gene for resistance. The consequences of relying on individual scoring occasions for studying Vf scab resistance are discussed in the context of linkage analysis, conventional breeding selection, and marker-assisted selection. Received: 23 July 1997 / Accepted: 31 October 1997