Qualitative alterations in plasma esterases in BALB/c mice following the administration of diethylnitrosamine.

Selected non-specific plasma esterases in female BALB/c mice, separated by polyacrylamide gel electrophoresis, were qualitatively altered following treatment of the mice with diethylnitrosamine (DEN). These alterations include a decreased preference for naphthyl butyrate as a substrate relative to naphthyl acetate; decreased sensitivity to enhancement by divalent cations such as Ca2+, Mg2+, and Mn2; and an increased sensitivity to inhibition by eserine. All esterase species affected were also quantitatively altered and some were testosterone-dependent.     

Opportunities to replace the use of animals in sepsis research. The report and recommendations of a Focus on Alternatives workshop

Sepsis and multiple organ failure are common causes of death in patients admitted to intensive care units. The incidence of sepsis and associated mortalities has been steadily increasing over the past 20 years. Sepsis is a complex inflammatory condition, the precise causes of which are still poorly understood. Animal models of sepsis have the potential to cause substantial suffering, and many of them have been poorly representative of the human syndrome. However, a number of non-animal approaches, including in vitro, in silico and clinical studies, show promise for addressing this situation. This report is based on discussions held at an expert workshop convened by Focus on Alternatives and held in 2004 at the Wellcome Trust, London. It provides an overview of some non-animal approaches to sepsis research, including their strengths and weaknesses, and argues that they should be prioritised for further development.     

A positive role for the Ku complex in DNA replication following strand break damage in mammals

Ku70-Ku80 complex is the regulatory subunit of DNA-dependent protein kinase (DNA-PK) and plays an essential role in double-strand break repair following ionizing radiation (IR). It preferentially interacts with chromosomal breaks and protects DNA ends from nuclease attack. Here we show evidence that cells defective in Ku80 exhibit a significantly slow S phase progression following DNA damage. IR-induced retardation in S phase progression in Ku80-/- cells was not due to the lack of DNA-PK kinase activity because both wild-type cells and DNA-PKcs-deficient cells showed no such symptom. Instead, proliferating cell nuclear antigen (PCNA) dissociated from chromosomes following IR in Ku80-deficient cells but not in wild-type or DNA-PKcs-deficient cells. Treatment of HeLa cells with IR induced colocalization of the Ku complex with PCNA on chromosomes. Together, these results suggest that binding of the Ku complex at chromosomal breaks may be necessary to maintain the sliding clamps (PCNA) on chromatin, which would allow cells to resume DNA replication without a major delay following IR.     

A role for the Fanconi anemia C protein in maintaining the DNA damage-induced G2 checkpoint

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.     

Pulmonary aspiration in preschool children with cystic fibrosis

Pulmonary aspiration of gastric refluxate (PAGR) has been demonstrated in association with pulmonary inflammation in school aged children with Cystic Fibrosis (CF). We sought to determine if similar findings were present in preschool children. Pepsin was measured in Broncho-alveolar lavage (BAL) fluid collected from clinically stable preschool children with CF and controls. Elevated pepsin levels were found in a subgroup of children with CF, but this was not found to be associated with pulmonary infection, pulmonary inflammation or respiratory or gastrointestinal symptoms.     

P32 distribution in phosphorus fractions of phosphorus-sensitive and -tolerant soybeans

Summary P³² incorporation into various phosphorylated compounds of roots, stems, and leaves was studied with P-sensitive (Clark) and P-tolerant (L9) varieties of soybeans. With increasing P concentration in the pretreatment solution, more P³² was incorporated into the inorganic phosphate fraction, mainly in which form P seemed to be transported from roots to leaves. P³² percentage of inorganic phosphate also showed a continuous decrease with absorption time. The rate of phosphorylation, as manifested by the ratio of P³² percentage or specific activities of the organic acid-soluble P to that of inorganic-P, was higher with the P-tolerant variety than with the P-sensitive one. The rate of RNA synthesis also seemed to be higher with the tolerant variety. Paper No. 7082, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Stability Analysis of a Model of Interaction Between the Immune System and Cancer Cells in Chronic Myelogenous Leukemia

We describe here a simple model for the interaction between leukemic cells and the autologous immune response in chronic phase chronic myelogenous leukemia (CML). This model is a simplified version of the model we proposed in Clapp et al. (Cancer Res 75:4053–4062, 2015). Our simplification is based on the observation that certain key characteristics of the dynamics of CML can be captured with a three-compartment model: two for the leukemic cells (stem cells and mature cells) and one for the immune response. We characterize the existence of steady states and their stability for generic forms of immunosuppressive effects of leukemic cells. We provide a complete co-dimension one bifurcation analysis. Our results show how clinical response to tyrosine kinase inhibitors treatment is compatible with the existence of a stable low disease, treatment-free steady state.     

Irradiated laboratory animal diets: Dominant lethal studies in the mouse

In 4 separate dominant lethal experiments groups of mice of either Charles River CD1 or Alderley Park strains were fed laboratory diets (Oakes, 41B, PRD, BP nutrition rat and mouse maintenance diet No. 1). The diets were either untreated (negative control diets) or irradiated at 1, 2.5 and 5 megarad and were freshly irradiated, or stored. The animals were fed their test diets for a period of 3 weeks prior to mating. Groups of mice given a single intraperitoneal injection of 200 mg cyclophosphamide per kg body weight served as the positive controls.

Freshly irradiated PRD diet fed to male mice of both strains caused an increase in early deaths in females mated to the males in week 7 and to a lesser extent in week 4. The increase due to irradiation was small by comparison with that produced by the positive control compound. The responses for the other irradiated diets showed no significant increases in early deaths although some values for Oakes diet were high. The effect of storage was examined with PRD and BPN diet on one occasion and produced conflicting results.

Thus there was some evidence that irradiated PRD diet has weak mutagenic activity in the meiotic and/or pre-meiotic phase of the spermatogenic cycle which appeared to be lessened on storage; the inclusion of such a diet in toxicological studies would therefore need to be carefully considered.     

Visualization and biopsy of the colon in tamarins and marmosets by endoscopy

Endoscopic visualization and biopsy have been performed under anesthesia in more than 65 tamarins and marmosets to study the pathogenesis of colitis and cancer of the colon. This procedure allows examination of the large bowel from the anus to the cecum and has been repeated at 2-6 month intervals with few complications. However, care must be exercised not to perforate the colon. Successful use of this technique will permit study of the pathogenesis of colonic diseases throughout the life of the animal and should provide cause-effect information about colitis and colon cancer in tamarins that may apply to the human diseases.