Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.LacI transgenic mice carry a recoverable λ phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of β-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin^-IL7R^-Sca-1⁺cKit⁺⁺(LSK) cells and other subpopulations of the hematopoietic system.     

Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b₅, and NADH-dependent cytochrome b₅ reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar K_cat and V_max values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.     

The effect of pregnancy on the interval between one oestrus and the next in the tammar wallaby, Macropus eugenii.

In females mated to vasectomized males the interval between oestrous periods was 30.6 +/- 1.17 (s.d.) days but when mated to intact males and pregnancy intervened, it was 29. +/- 1.26 days (P less than 0.0025). After removal of pouch young, females carrying diapausing blastocysts gave birth 26.2 days later and came into oestrus 26.4 +/- 0.57 (s.d.) days later. When post-partum fertilization was prevented, removal of pouch young was followed by oestrus 30.4 +/- 0.99 days later (P less than 0.0005). These results indicate an influence of the conceptus upon the oestrous cycle of Macropus eugenii.     

Amino acid utilization by L-M strain mouse cells in a chemically defined medium

Summary The amino acid requirements of strain L-M mouse cells grown in a chemically defined medium (2×Eagle) containing only the 13 essential amino acids (EAA) were investigated. Medium and acid hydrolysate samples were analyzed for amino acid content by the method of ion exchange chromatography. The extent of utilization of the EAA differed;e.g. after 120 hr of cell growth without medium change, glutamine was exhausted from the medium; methionine, leucine, isoleucine, cystine, arginine, and valine were depleted 60 to 80%; other EAA were used to lesser extents. Although the EAA were used in excess of their requirements for protein synthesis, a correlation could generally be made between utilization and protein amino acid composition. Glutamine appeared to be, a growth-limiting factor. Use of U-¹⁴C-labeled glutamine indicated that over one-half of the metabolized glutamine was converted to carbon dioxide, 17% to cell material, and 15% was extracted from the amino acid pools. Nonessential amino acids (NEAA), viz. alanine, aspartic acid, glutamic acid, glycine, proline, and serine, were released into the medium during growth, and some were reutilized. Exogenous provision of these did not improve cell growth. In contrast to the other NEAA, only serine showed net utilization when provided exogenously. When glutamic acid largely replaced the glutamine in the medium, it exerted a sparing effect on the glutamine requirement for protein synthesis. Suggestions are given for the improvement of Eagle medium for cell growth. Supported by Research Grants CA 03720 and CA 11802 from the National Institutes of Health. Predoctoral, fellow supported, by Grant F01-GM-42156-02 from the National Institutes of Health.     

Network quantification of EGFR signaling unveils potential for targeted combination therapy

The epidermal growth factor receptor (EGFR) signaling network is activated in most solid tumors, and small‐molecule drugs targeting this network are increasingly available. However, often only specific combinations of inhibitors are effective. Therefore, the prediction of potent combinatorial treatments is a major challenge in targeted cancer therapy. In this study, we demonstrate how a model‐based evaluation of signaling data can assist in finding the most suitable treatment combination. We generated a perturbation data set by monitoring the response of RAS/PI3K signaling to combined stimulations and inhibitions in a panel of colorectal cancer cell lines, which we analyzed using mathematical models. We detected that a negative feedback involving EGFR mediates strong cross talk from ERK to AKT. Consequently, when inhibiting MAPK, AKT activity is increased in an EGFR‐dependent manner. Using the model, we predict that in contrast to single inhibition, combined inactivation of MEK and EGFR could inactivate both endpoints of RAS, ERK and AKT. We further could demonstrate that this combination blocked cell growth in BRAF‐ as well as KRAS‐mutated tumor cells, which we confirmed using a xenograft model.     

VP4-specific intestinal antibody response to rotavirus in a murine model of heterotypic infection.

We have adapted a murine model of heterotypic rotavirus infection for the purpose of evaluating the intestinal antibody response to an infection that mimics human vaccination. Neonatal mice were infected with the rhesus rotavirus (RRV). The enzyme-linked immunospot assay was used in order to avoid common artifacts in the quantitation of intestinal immune responses inherent in measurements of luminal or serum immunoglobulins and to obtain easily quantifiable data in a flexible and convenient format. Functionally active lymphocytes were harvested from the spleen, small intestinal lamina propria, Peyer's patches, and mesenteric lymph nodes and processed into single-cell suspensions. Antibody-secreting cells (ASC) were quantitated from 5 to 50 days after infection for total, RRV-specific, baculovirus-expressed VP4-specific, and single-shell RRV-specific ASC secreting either immunoglobulin G (IgG), IgM, or IgA. The response to VP4 constituted less than 1.5% of the total virus-specific response, which was located almost exclusively in the gut and was 90% IgA. Intestinal ASC were directed overwhelmingly toward proteins incorporated in the single-shell particle, predominantly VP2 and VP6. We conclude that the antibody response to VP4, thought to be the site of the important neutralization sites conserved among several rotavirus serotypes, is an extremely small portion of the overall antibody response in the intestinal tract.