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.     

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.     

Reconstitution of the NF1 GAP-related domain in NF1-deficient human Schwann cells

Schwann cells derived from peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are deficient for the protein neurofibromin, which contains a GAP-related domain (NF1-GRD). Neurofibromin-deficient Schwann cells have increased Ras activation, increased proliferation in response to certain growth stimuli, increased angiogenic potential, and altered cell morphology. This study examined whether expression of functional NF1-GRD can reverse the transformed phenotype of neurofibromin-deficient Schwann cells from both benign and malignant peripheral nerve sheath tumors. We reconstituted the NF1-GRD using retroviral transduction and examined the effects on cell morphology, growth potential, and angiogenic potential. NF1-GRD reconstitution resulted in morphologic changes, a 16-33% reduction in Ras activation, and a 53% decrease in proliferation in neurofibromin-deficient Schwann cells. However, NF1-GRD reconstitution was not sufficient to decrease the in vitro angiogenic potential of the cells. This study demonstrates that reconstitution of the NF1-GRD can at least partially reverse the transformation of human NF1 tumor-derived Schwann cells.     

Capping of CdSe-ZnS quantum dots with DHLA and subsequent conjugation with proteins

We provide a detailed protocol for designing water-soluble CdSe-ZnS quantum dots (QDs) based on cap exchange of the native hydrophobic shell with dihydrolipoic acid (DHLA) ligands, and the preparation of functional QD bioconjugates for use in immunoassays. Our conjugation strategy is based on non-covalent self-assembly between DHLA-capped QDs and protein appended with either an electrostatic attachment domain (namely, the basic leucine zipper) or a polyhistidine tag. These bioconjugates combine the properties of the QD and attached biomolecule to create structures with desirable luminescent and biologically specific properties. This method also allows the preparation of mixed surface conjugates, which results in the conjugates gaining multiple biological activities. Conjugation of DHLA-capped QDs to maltose binding protein (MBP), the immunoglobulin-G-binding beta2 domain of streptococcal protein G (PG) and avidin will be described. MBP and PG were modified by genetic fusion with either a charged leucine zipper or a polyhistidine interaction domain.