MUC4 involvement in ErbB2/ErbB3 phosphorylation and signaling in response to airway cell mechanical injury

The receptor tyrosine kinases ErbB2 and ErbB3 are phosphorylated in response to injury of the airway epithelium. Since we have shown that the membrane mucin MUC4 can act as a ligand/modulator for ErbB2, affecting its localization in polarized epithelial cells and its phosphorylation, we questioned whether Muc4 was involved, along with ErbB2 and ErbB3, in the damage response of airway epithelia. To test this hypothesis, we first examined the localization of MUC4 in human airway samples. Both immunocytochemistry and immunofluorescence showed a co‐localization of MUC4 and ErbB2 at the airway luminal surface. Sequential immunoprecipitation and immunoblotting from airway cells demonstrated that the MUC4 and ErbB2 are present as a complex in airway epithelial cells. To assess the participation of MUC4 in the damage response, cultures of NCI‐H292 or airway cells were scratch‐wounded, then analyzed for association of phospho‐ErbB2 and ‐ErbB3 with MUC4 by sequential immunoprecipitation and immunoblotting. Wounded cultures exhibited increased phosphorylation of both receptors in complex with MUC4. Scratch wounding also increased activation of the downstream pathway through Akt, as predicted from our previous studies on Muc4 effects on ErbB2 and ErbB3. The participation of MUC4 in the phosphorylation response was also indicated by siRNA repression of MUC4 expression, which resulted in diminution of the phosphorylation of ErbB2 and ErbB3. These studies provide a new model for the airway epithelial damage response, in which the MUC4–ErbB2 complex is a key element in the sensor mechanism and phosphorylation of the receptors. J. Cell. Biochem. 107: 112–122, 2009. © 2009 Wiley‐Liss, Inc.     

Definition and Estimation of Higher-Order Gene Fixation Indices

Fixation indices summarize the associations between genes that arise from the joint effects of inbreeding and selection. In this paper, fixation indices are derived for pairs, triplets and quadruplets of genes at a single multiallelic locus. The fixation indices are obtained by dividing cumulants by constants; the cumulants describe the statistical distribution of alleles and the constants are functions of gene frequency. The use of cumulants instead of moments is necessary only for four-gene indices, when the fourth cumulant is used. A second type of four-gene index is also required, and this index is based upon the covariation of second-order cumulants. At multiallelic loci, a large number of indices is possible. If alleles are selectively neutral, the number of indices is reduced and the relationship between gene identity and gene cumulants is shown.--Two-gene indices can always be estimated from genotypic frequency data at a single polymorphic locus. Three-gene indices are also estimable except when allele frequency equals one-half. Four-gene indices are not estimable unless selection is assumed to have an equal effect upon each allele (such as under selective neutrality) and the locus contains at least three alleles of unequal frequency. For diallelic or selected loci, an alternative four-gene fixation index is proposed. This index incorporates both types of four-gene associations but cannot be related to gene identity.     

Cellulose and lignin degradation in forest soils: Response to moisture,temperature, and acidity

The concentration of lignin in plant tissue is a major factor controlling organic matter degradation rates in forest ecosystems. Microbial biomass and lignin and cellulose decomposition were measured for six weeks in forest soil microcosms in order to determine the influence of pH, moisture, and temperature on organic matter decomposition. Microbial biomass was determined by chloroform fumigation; lignin and cellulose decomposition were measured radiometrically. The experiment was designed as a Latin square with soils of pH of 4.5, 5.5, and 6.5 adjusted to 20, 40, or 60% moisture content, and incubated at temperatures of 4, 12, or 24°C. Microbial biomass and lignin and cellulose decomposition were not significantly affected by soil acidity. Microbial biomass was greater at higher soil moisture contents. Lignin and cellulose decomposition significantly increased at higher soil temperatures and moisture contents. Soil moisture was more important in affecting microbial biomass than either soil temperature or soil pH.     

A MARKER-BASED METHOD FOR INFERENCES ABOUT QUANTITATIVE INHERITANCE IN NATURAL POPULATIONS

A marker-based method for studying quantitative genetic characters in natural populations is presented and evaluated. The method involves regressing quantitative trait similarity on marker-estimated relatedness between individuals. A procedure is first given for estimating the narrow sense heritability and additive genetic correlations among traits, incorporating shared environments. Estimation of the actual variance of relatedness is required for heritability, but not for genetic correlations. The approach is then extended to include isolation by distance of environments, dominance, and shared levels of inbreeding. Investigations of statistical properties show that good estimates do not require great marker polymorphism, but rather require significant variation of actual relatedness; optimal allocation generally favors sampling many individuals at the expense of assaying fewer marker loci; when relatedness declines with physical distance, it is optimal to restrict comparisons to within a certain distance; the power to estimate shared environments and inbreeding effects is reasonable, but estimates of dominance variance may be difficult under certain patterns of relationship; and any linkage of markers to quantitative trait loci does not cause significant problems. This marker-based method makes possible studies with long-lived organisms or with organisms difficult to culture, and opens the possibility that quantitative trait expression in natural environments can be analyzed in an unmanipulative way.     

INFERENCES ABOUT QUANTITATIVE INHERITANCE BASED ON NATURAL POPULATION STRUCTURE IN THE YELLOW MONKEYFLOWER,MIMULUS GUTTATUS

We used a nonmanipulative, marker-based method to study quantitative genetic inheritance in two habitats of a common monkeyflower population. The method involved regressing quantitative trait similarity on marker-estimated relatedness between individuals sampled in the field. We sampled 300 adult plants from each of two transects, one along a stream habitat and another through a meadow habitat. For each plant we measured 10 quantitative characters and assayed 10 polymorphic isozyme loci. In the meadow habitat, relatedness of plants within 1 m was moderate (r = 0.125, corresponding to half-sibs) as was actual variance of relatedness (V_r = 0.044). Significant heritabilities of 50–70% were found for corolla width and the fitness characters of flower number and plant weight. Genetic correlations were strongly positive, but sharing of environmental effects within 1 m was weak. In the stream habitat, levels of relatedness were lower and similar heritabilities were indicated. To detect dominance variance and the correlation of phenotypes due to shared inbreeding, we also estimated higher-order coefficients of relationship and inbreeding, but these did not significantly differ from zero. Laboratory-based estimates of heritability in the field were lower than the marker-based estimates, indicating that natural heritabilities and genetic correlations may be stronger than indicated by controlled studies.     

INBREEDING DEPRESSION IN TWO MIMULUS TAXA MEASURED BY MULTIGENERATIONAL CHANGES IN THE INBREEDING COEFFICIENT

In mixed-mating plant populations, one can estimate the relative fitness of selfed progeny w by measuring the inbreeding coefficient F and selfing rate s of adults of one generation, together with F of adults in the following generation (after selection). In the first application of this multigenerational method, we estimated F and s for adults over three consecutive generations in adjacent populations of two annual Mimulus taxa: the outbreeding M. guttatus and the inbreeding M. platycalyx. This gave estimates of w for the last two generations. Although average multilocus selfing rates were high in both taxa (0.63 in M. guttatus; 0.84 in M. platycalyx), the relative fitness of selfed progeny averaged only 0.19 in M. guttatus and 0.32 in M. platycalyx. An alternative estimator for w that incorporates biparental inbreeding gave even lower estimates of w. These values are significantly below the 0.5 threshold thought to favor selfing, and show that partially selfing populations can harbor substantial genetic load. In accordance with the purging hypothesis, the more highly selfing M. platycalyx showed marginally lower inbreeding depression than M. guttatus in both years (P = 0.08). Inbreeding depression and selfing rates also varied among years in concert among taxa. Several sources of bias are discussed, but computer simulations indicate it is unlikely that w is biased downwards by linkage of marker loci to load loci.     

MULTIPLE PATERNITY AND SELF-FERTILIZATION IN RELATION TO FLORAL AGE IN MIMULUS GUTTATUS (SCROPHULARIACEAE)

To gain some understanding of the mechanisms responsible for the intermediate levels of both multiple paternity and selfing observed previously in the common monkeyflower, Mimulus guttatus, we performed a field experiment to assess the role of 1) multiple paternity through sequential pollinator visits and 2) selfing through corolla abscission. In M. guttatus, flowers remain open for several days; then, in the process of corolla abscission anthers drag past the stigma. We predicted that multiple visitation by pollinators over this period should increase the degree of multiple paternity of sibling arrays, and that corolla dragging may be the primary cause of the observed selfing. In three northern California populations corollas were removed after being open for 1 day (preventing further mating), and the type of matings resulting was compared to matings from adjacent flowers with undisturbed corollas. On average, seed set was twice as great in flowers with unmanipulated corollas, indicating that about half of all matings in the lifespan of a M. guttatus flower occur after 1 day. Pollen supplementation revealed that pollen was limiting in one population. Electrophoretic assay of progeny showed multiple paternity was greater in unmanipulated flowers in the two populations that were not pollen limited, suggesting that sequential pollination may be an important source of multiple paternity. However, no difference was detected in the selfing rate between the two treatments, suggesting that corolla dragging was not a source of selfing even under pollen-limited conditions.     

Isolation, characterization, and cross-species utility of microsatellites in yellow cedar (Chamaecyparis nootkatensis).

Chamaecyparis nootkatensis is an ecologically and economically important conifer of the north Pacific coastal forests. To aid in studies of clonal structure and genetic differentiation of this and related species, we isolated and characterized microsatellites from C. nootkatensis. A microsatellite-enriched library yielded 75 repeat-containing sequences for which primer pairs were designed. Only five showed reliable amplification and polymorphism, with an average of 13.7 alleles/locus and a mean expected heterozygosity of 0.592. In progeny tests with four families, few null alleles were directly detected and loci segregated according to Mendelian expectations. However, in one primer pair, high heterozygote deficiency was observed, suggesting the presence of a null allele. The ability of primer pairs to cross amplify was tested on 18 species of the Cupressaceae sensu lato; three primer pairs yielded polymorphic loci in Cupressus and Juniperus species, but not in other Chamaecyparis species. This also supports recent findings of a closer affinity of C. nootkatensis with Cupressus over other Chamaecyparis species.     

Detection of low molecular weight copper(II) and zinc(II) binding ligands in ultrafiltered milks—the citrate connection

Low molecular weight zinc(II) and copper(II) binding ligands were detected in ultrafiltered human, bovine, and goat milk by the application of the method of modified gel chromatography. Human milk contains at least three detectable low molecular weight copper binders, whereas bovine and goat milk contain at least two. All three milks show two copper binding peaks with the same elution volumes. Zinc chromatograms were less specific than copper. Zinc showed only a single detectable low molecular weight binding ligand common to all three milks. Elution volumes for both zinc(II) and copper(II) citrate and picolinate systems were measured. Elution volumes of both copper(II) and zinc(II) citrate complexes are identical to elution volumes of an intense peak observed with all three milks; it is reasonable to assume that at least part of this peak corresponds to citrate. Human milk alone has a relatively intense binding peak for copper(II) at the same elution volume as the glutamate complex. Human and goat milk have another low intensity copper(II) binding ligand peak at the same elution volume; a number of amino acid complexes have binding peaks at this position. No peak characteristic of the zinc(II) or copper(II) picolinate systems could be found with any of the milks.     

INFERENCES ABOUT INBREEDING DEPRESSION BASED ON CHANGES OF THE INBREEDING COEFFICIENT

In a mixed-mating population, the fitness of selfed individuals, relative to outcrossed individuals, can be estimated from observed changes of the inbreeding coefficient F. In general, three measurements of F, or two measurements of F and one of the selfing rate, spanning two generations are needed. If however, adult F is assumed constant among generations, the adult F and selfing rate of one generation are sufficient to estimate relative fitness. Estimates of the relative fitness of selfed individuals for 14 Mimulus populations, assuming equilibrium of adult F, averaged 0.38, which is significantly lower than the 0.50 threshold needed to favor selfing genotypes. However, estimates for some populations showed large variance and over the 14 populations, relative fitness did not correlate with selfing rate. Violations of the equilibrium assumption causes a positive bias of the estimate and a spurious negative correlation of estimates with selfing rate. Estimates of relative fitness based on two generations of F do not suffer these problems. The need for large sample sizes, multiallelic loci, and moderate levels of natural selfing limits the usefulness of using changes of F to infer inbreeding depression.