Factors influencing the growth of alveolar type II epithelial cells isolated from rat lungs

Primary cultures of isolated alveolar type II cells have been established. Under appropriate conditions, these epithelial cells can be subcultured at least nine times. Using standard assay procedures, effects of growth factors or inhibitors can be studied. The alveolar type II cells show a marked response to both serum and growth factors in tissue culture. Either epidermal growth factor (EGF) or human urine gives an increase in thymidine incorporation (2-fold and 10-fold, respectively). The growth factor(s) in urine appears to be different from urogastrone (human EGF). The response to urine is several-fold greater than the response to a saturating concentration of mouse EGF alone. Mouse EGF added to urine does not increase the activity of urine. The period during which the alveolar type II cells respond to the growth factor(s) in urine is limited to early passages of the cells. Alveolar type II epithelial cells produce growth inhibitors in culture. Inhibitors are produced in the growth medium in either the presence or absence of serum. The concentrated inhibitor, although very unstable, gives up to a 50% inhibition of thymidine incorporation when assayed on sparse or crowded alveolar type II cells.     

Mapping of QTLs for lateral and axile root growth of tropical maize

Maize genotypes may adapt to dry environments by avoiding desiccation by means of a deeper root system or by maintaining growth and water extraction at low water potentials. The aim of this study was to determine the quantitative genetic control of root growth and root morphology in a population of 236 recombinant inbred lines (RILs) from the cross between CML444 (high-yielding) × SC-Malawi (low-yielding), which segregates for the response to drought stress at flowering. The RILs and the parental lines were grown on blotting paper in growth pouches until the two-leaf stage under non-stressed conditions; the parents were additionally exposed to desiccation stress induced by polyethylene glycol with a molecular weight of 8000 Dalton (PEG-8000). The lengths of axile and lateral roots were measured non-destructively at 2, 5, 7 and 9 days after germination, by scanning with an A4 scanner followed by digital image analysis. CML444 had a lower rate constant of lateral root elongation (k_Lat) than SC-Malawi, but the two genotypes did not differ in their response to desiccation. QTLs affecting root vigor, as depicted by increments in k_Lat, the elongation rate of axile roots (ER_Ax) and the number of axile roots (No_Ax) were identified in bins 2.04 and 2.05. QTLs for No_Ax and ER_Ax collocated with QTLs for yield parameters in bins 1.03–1.04 and 7.03–04. The correspondence of QTLs for axile root traits in bins 1.02–1.03 and 1.08 and QTLs for lateral roots traits in bins 2.04–2.07 in several mapping populations suggests the presence of genes controlling root growth in a wide range of genetic backgrounds.     

Interaction of the antileukemic alkaloid, 2-hydroxy-3,8,9-trimethoxy-5-methylbenzo[c]phenanthridine (fagaronine), with nucleic acids

The novel alkaloid 2-hydroxy-3,8,9-trimethoxy-5-methylbenzo[c]phenanthridine was isolated from the roots of Fagara zanthoxyloides, an indigenous plant of Ghana, structurally characterized and assigned the trivial name fagaronine. Fagaronine is a potent antileukemic agent. We presently report a detailed isolation procedure and the results of studies designed to examine its mechanism of action. Admixture of calf thymus DNA produced a hypsochromic shift in the absorption spectra of fagaronine, with a remarkable increase in intensity in the range of 400 nm. The extent of the spectral alterations were dependent on the concentration of DNA which was added, and clear isosbestic points, indicative of a single mode of interaction, were generated. As determined by gel filtration column chromatography, the DNA-fagaronine complex formed with an association constant of 4.7 × 10⁴ M^?1 and the maximum extent of binding was 1.0 fagaronine molecule per 4.5 base pairs. Contrary to previous literature reports, the binding of fagaronine was not limited to regions of DNA containing adenine and thymine. Isosbestic points were observed following admixture with poly-(dA-dT), as well as poly(dG) · poly(dC). The most likely mode of interaction is intercalation. As judged by spectral titrations, fagaronine could also interact with ribonucleic acids. As compared with single-stranded homopolymers [e.g., poly(A), poly(U)], the interaction with double-stranded species [e.g., poly(A) · poly(U), poly(I) · poly(C)] was more extensive. Although the spectra differed from those obtained with deoxyribonucleic acids, single isosbestic points could be generated. Additionally, utilizing Salmonella typhimurium strain TM677, fagaronine was found to be bactericidal but not mutagenic. The bactericidal activity was enhanced about 1000-fold in the presence of a 9000 × g supernatant fraction derived from rat liver and the activating component was further characterized as being heat labile and localized in the microsomal subfraction. Preliminary investigations did not support the notion that fagaronine was metabolized to an active species responsible for this effect. When KB cells were treated with fagaronine, it was also found to be cytotoxic. In contrast, N-demethylfagaronine was not bactericidal or cytotoxic, nor could it effectively interact with DNA. Thus, the cationic (quaternary) nitrogen atom of fagaronine is of requisite importance, and a key mechanism of cytotoxic activity may be interaction with cellular nucleic acids.     

Using gas chromatography to determine winter diets of greater sage-grouse in Utah

Sagebrush (Artemisia spp.) constitutes the majority (>99%) of sage-grouse (Centrocercus spp.) winter diets. Thus, identification and protection of important winter habitats is a conservation priority. However, not all sagebrush may be alike. More information is needed regarding sage-grouse sagebrush winter dietary preferences for application to management. The objective of our research was to determine if chemical analysis of fecal pellets could be used to characterize winter sage-grouse diets as a substitute for more invasive methods. We collected and analyzed fecal pellets and sagebrush samples from 29 different sage-grouse flock locations in northwestern and southcentral Utah. Using gas chromatography, we were able to identify crude terpene profiles that were unique to Wyoming sagebrush (A. tridentata wyomingensis) and black sagebrush (A. nova). We subsequently used the profiles to determine sagebrush composition of sage-grouse fecal pellets, thus reflecting sage-grouse winter diets. This technique provides managers with a tool to determine which species or subspecies of sagebrush may be important in the winter diets of sage-grouse populations. © 2011 The Wildlife Society.     

Direct monitoring of capillary perfusion following normovolemic hemodilution in an experimental skin-flap model

The effects of normovolemic hemodilution on skin flap survival are studied in a recently developed skin-flap model (homozygous hairless mouse ear) in which nutritional capillary flow is monitored directly by means of intravital microscopy from the time of flap creation throughout the establishment of necrosis. Two diluting agents (dextran 60 and hydroxyethyl starch 200) are utilized. Our quantitative findings demonstrate that the amount of nonperfused tissue following flap creation in both the dextran (n = 23) and starch (n = 13) groups was significantly decreased as compared with controls (n = 19). Our qualitative observations suggest that improved hemorrheologic properties at the microcirculatory level are responsible for the observed decreased necrosis. Various mechanisms by which hemodilution may act to prevent necrosis are discussed.     

Identification of factors involved in target RNA-directed microRNA degradation

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3′-5′ exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.