ATM and the catalytic subunit of DNA-dependent protein kinase activate NF-kappaB through a common MEK/extracellular signal-regulated kinase/p90(rsk) signaling pathway in response to distinct forms of DNA damage

We have identified a novel pathway of ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) signaling that results in nuclear factor kappaB (NF-kappaB) activation and chemoresistance in response to DNA damage. We show that the anthracycline doxorubicin (DOX) and its congener N-benzyladriamycin (AD 288) selectively activate ATM and DNA-PK, respectively. Both ATM and DNA-PK promote sequential activation of the mitogen-activated protein kinase (MAPK)/p90(rsk) signaling cascade in a p53-independent fashion. In turn, p90(rsk) interacts with the IkappaB kinase 2 (IKK-2) catalytic subunit of IKK, thereby inducing NF-kappaB activity and cell survival. Collectively, our findings suggest that distinct members of the phosphatidylinositol kinase family activate a common prosurvival MAPK/IKK/NF-kappaB pathway that opposes the apoptotic response following DNA damage.     

Death and renal transplantation among Aboriginal people undergoing dialysis

Background

Despite the increase in the number of Aboriginal people with end-stage renal disease around the world, little is known about their health outcomes when undergoing renal replacement therapy. We evaluated differences in survival and rate of renal transplantation among Aboriginal and white patients after initiation of dialysis.

Methods

Adult patients who were Aboriginal or white and who commenced dialysis in Alberta, Saskatchewan or Manitoba between Jan. 1, 1990, and Dec. 31, 2000, were recruited for the study and were followed until death, transplantation, loss to follow-up or the end of the study (Dec. 31, 2001). We used Cox proportional hazards models to examine the effect of race on patient survival and likelihood of transplant, with adjustment for potential confounders.

Results

Of the 4333 adults who commenced dialysis during the study period, 15.8% were Aboriginal and 72.4% were white. Unadjusted rates of death per 1000 patient-years during the study period were 158 (95% confidence interval [CI] 144–176) for Aboriginal patients and 146 (95% CI 139–153) for white patients. When follow-up was censored at the time of transplantation, the age-adjusted risk of death after initiation of dialysis was significantly higher among Aboriginal patients than among white patients (hazard ratio [HR] 1.15, 95% CI 1.02–1.30). The greater risk of death associated with Aboriginal race was no longer observed after adjustment for diabetes mellitus and other comorbid conditions (adjusted HR 0.89, 95% CI 0.77–1.02) and did not appear to be associated with socioeconomic status. During the study period, unadjusted transplantation rates per 1000 patient-years were 62 (95% CI 52–75) for Aboriginal patients and 133 (95% CI 125–142) for white patients. Aboriginal patients were significantly less likely to receive a renal transplant after commencing dialysis, even after adjustment for potential confounders (HR 0.43, 95% CI 0.35–0.53). In an additional analysis that included follow-up after transplantation for those who received renal allografts, the age-adjusted risk of death associated with Aboriginal race (HR 1.36, 95% CI 1.21–1.52) was higher than when follow-up after transplantation was not considered, perhaps because of the lower rate of transplantation among Aboriginals.

Interpretation

Survival among dialysis patients was similar for Aboriginal and white patients after adjustment for comorbidity. However, despite universal access to health care, Aboriginal people had a significantly lower rate of renal transplantation, which might have adversely affected their survival when receiving renal replacement therapy.In North America and the Antipodes, the incidence of diabetes among adolescent and adult Aboriginals has risen dramatically,^1,2,3,4 with corresponding increases in the prevalence of diabetic nephropathy.^5,6,7 Aboriginal people in Canada have experienced disproportionately high incidence rates of end-stage renal disease (ESRD), with an 8-fold increase in the number of prevalent dialysis patients between 1980 and 2000.⁸ Although the incidence of ESRD appears to have decreased in recent years, the prevalence of diabetes mellitus and its complications are rising, especially among young people.^9,10,11Most work evaluating health outcomes among Aboriginal people considers either the general population¹²or diseases for which interventions are implemented over a short period, such as alcohol abuse,¹³ injury¹⁴ or critical illness.¹⁵ Death and markers of poor health are significantly more common among Aboriginal people than among North Americans of European ancestry, perhaps because of the greater prevalence of diabetes mellitus, adverse health effects due to lower socioeconomic status¹⁶ and reduced access to primary care.¹⁷ Aboriginal patients may also face unique barriers to care, including mistrust of non-Aboriginal providers, institutional discrimination or preference for traditional remedies.¹⁸ These factors may be most relevant when contact with physicians is infrequent, which obstructs development of a therapeutic relationship. In contrast, ESRD is a chronic illness that requires ongoing care from a relatively small, stable multidisciplinary team.Although recent evidence highlights racial inequalities in morbidity and mortality among North Americans with ESRD, most studies have focused on black or Hispanic populations.¹⁹We conducted this study to evaluate rates of death and renal transplantation among Aboriginal people after initiation of dialysis in Alberta, Saskatchewan and Manitoba.     

The human immunodeficiency virus (HIV) protease inhibitor indinavir directly affects the opportunistic fungal pathogen Cryptococcus neoformans

Highly active antiretroviral therapy (HAART), that includes human immunodeficiency virus (HIV) protease inhibitors (PIs), has been remarkably efficacious including against some opportunistic infections. In this report we investigated the effect(s) of the PI indinavir on protease activity by Cryptococcus neoformans, an opportunistic fungal pathogen responsible for recurrent meningoencephalitis in AIDS patients. Indinavir was also tested for potential effects on other parameters, such as fungal viability, growth ability and susceptibility to immune effector cells. It was found that indinavir impaired cryptococcal protease activity in a time- and dose-dependent fashion. The phenomenon was similarly detectable in ATCC/laboratory strains and clinical isolates. C. neoformans growth rate was also significantly reduced upon exposure to indinavir, while fungal viability was not affected and mitochondrial toxicity not detected. Furthermore, as assessed by an in vitro infection model, indinavir significantly and consistently augmented C. neoformans susceptibility to microglial cell-mediated phagocytosis and killing. Overall, by providing the first evidence that indinavir directly affects C. neoformans, these data add new in vitro insights on the wide-spectrum efficacy of PIs, further arguing for the clinical relevance of HAART against opportunistic infections in AIDS.     

Ezrin is essential for epithelial organization and villus morphogenesis in the developing intestine

Ezrin, Radixin, and Moesin (the ERM proteins) supply regulated linkage between membrane proteins and the actin cytoskeleton. The study of mammalian ERM proteins has been hampered by presumed functional overlap. We have found that Ezrin, the only ERM detected in epithelial cells of the developing intestine, provides an essential role in configuring the mouse intestinal epithelium. Surprisingly, Ezrin is not absolutely required for the formation of brush border microvilli or for the establishment or maintenance of epithelial polarity. Instead, Ezrin organizes the apical terminal web region, which is critical for the poorly understood process of de novo lumen formation and expansion during villus morphogenesis. Our data also suggest that Ezrin controls the localization and/or function of certain apical membrane proteins that support normal intestinal function. These in vivo studies highlight the critical function of Ezrin in the formation of a multicellular epithelium rather than an individual epithelial cell.     

Cre-mediated germline mosaicism: a new transgenic mouse for the selective removal of residual markers from tri-lox conditional alleles

The binary Cre-lox conditional knockout system requires an essential part of the target gene to be flanked by loxP sites, enabling excision in vivo upon Cre expression. LoxP sites are introduced by homologous recombination, together with a selectable marker. However, this marker can disturb gene expression and should be removed. The marker is therefore often prepared with a third, flanking loxP site (tri-lox construct), facilitating its selective removal by partial Cre-lox recombination. We have shown that this excision can be achieved in vivo in the germline using EIIaCre transgenic mice, and have described the advantages of in vivo over in vitro removal. We show here that MeuCre40, a new transgenic mouse, more reliably and reproducibly generates an optimal partial mosaic Cre-lox recombination pattern in the early embryo. This mosaicism was transmitted to the germline and to many other tissues. Alleles with partial deletions, in particular floxed alleles from which the selectable marker was removed, were readily recovered in the next generation, after segregation from the transgene. Segregation via paternal or maternal transmission led to successful recovery of the alleles of interest. We also obtained total deletion of the floxed regions in the same experiment, making this transgene a polyvalent Cre-lox tool. We rigorously tested the ability of MeuCre40 to solve tri-lox problems, by using it for the in vivo removal of neo^R- and hprt-expression cassettes from three different tri-lox mutants.     

Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes. Results of an international slide-scoring exercise by the HUMN project

One of the objectives of the HUman MicroNucleus (HUMN) project is to identify the methodological variables that have an important impact on micronucleus (MN) or micronucleated (MNed) cell frequencies measured in human lymphocytes using the cytokinesis-block micronucleus assay. In a previous study we had shown that the scoring criteria used were likely to be an important variable. To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with a total of 51 slide scorers involved. The results of this study show that even under these optimized conditions there is a great variation in the MN frequency or MNed cell frequency obtained by individual laboratories and scorers. All laboratories ranked correctly the MNed cell frequency in cells from cultures that were unirradiated, or exposed to 1 or 2Gy of gamma rays. The study also estimated that the intra-scorer median coefficient of variation for duplicate MNed cell frequency scores is 29% for unexposed cultures and 14 and 11% for cells exposed to 1 and 2Gy, respectively. These values can be used as a standard for quality or acceptability of data in future studies. Using a Poisson regression model it was estimated that radiation dose explained 67% of the variance, while staining method, cell sample, laboratory, and covariance explained 0.6, 0.3, 6.5, and 25.6% of the variance, respectively, leaving only 3.1% of the variance unexplained. As part of this exercise, nucleoplasmic bridges were also estimated by the laboratories; however, inexperience in the use of this biomarker of chromosome rearrangement was reflected in the much greater heterogeneity in the data and the unexplained variation estimated by the Poisson model. The results of these studies indicate clearly that even after standardizing culture and scoring conditions it will be necessary to calibrate scorers and laboratories if MN, MNed cell and nucleoplasmic bridge frequencies are to be reliably compared among laboratories and among populations.     

Organic tissues in rotating bioreactors: fluid-mechanical aspects, dynamic growth models, and morphological evolution

This analysis deals with advances in tissue-engineering models and computational methods as well as with novel results on the relative importance of "controlling forces" in the growth of organic constructs. Specifically, attention is focused on the rotary culture system, because this technique has proven to be the most practical solution for providing a suitable culture environment supporting three-dimensional tissue assemblies. From a numerical point of view, the growing biological specimen gives rise to a moving boundary problem. A "volume-of-fraction" method is specifically and carefully developed according to the complex properties and mechanisms of organic tissue growth and, in particular, taking into account the sensitivity of the construct/liquid interface to the effect of the fluid-dynamic shear stress (it induces changes in tissue metabolism and function that elicit a physiological response from the biological cells). The present study uses available data to introduce a set of growth models. The surface conditions are coupled to the transfer of mass and momentum at the specimen/culture-medium interface and lead to the introduction of a group of differential equations for the nutrient concentration around the sample and for the evolution of tissue mass displacement. The models are then used to show how the proposed surface kinetic laws can predict (through sophisticated numerical simulations) many of the known characteristics of biological tissues grown using rotating-wall perfused vessel bioreactors. This procedure provides a validation of the models and associated numerical method and also gives insight into the mechanisms of the phenomena. The interplay between the increasing size of the tissue and the structure of the convective field is investigated. It is shown that this interaction is essential in determining the time evolution of the tissue shape. The size of the growing specimen plays a critical role with regard to the intensity of convection and the related shear stresses. Convective effects, in turn, are found to impact growth rates, tissue size, and morphology, as well as the mechanisms driving growth. The method exhibits novel capabilities to predict and elucidate experimental observations and to identify cause-and-effect relationships.     

Increased acetylcholinesterase activities in specimens of Sparus auratus exposed to sublethal copper concentrations

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as V(max) and K(m) determination were carried out in tissue extracts of Cu-exposed or control animals. V(max) and K(m) were also determined with or without addition of Cu(2+) in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu(2+) addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis.