Aortic carboxypeptidase-like protein is expressed in collagen-rich tissues during mouse embryonic development

Aortic carboxypeptidase-like protein (ACLP) was originally identified in vascular smooth muscle cells and contains discoidin and catalytically inactive metallocarboxypeptidase domains. ACLP is a secreted protein that associates with the extracellular matrix and is essential for abdominal wall development and contributes to dermal wound healing. Because of these developmental and adult phenotypes, we examined the expression of ACLP by immunohistochemistry throughout mouse embryonic development. ACLP was not detected in 7.5 days post-coitum (dpc) embryos, however at 9.5 dpc low levels of expression were detected in the somites and dorsal aorta. Expression was detected in both the yolk sac and embryonic vasculature at 10.5d pc. ACLP expression increased in both large and small blood vessels at 11.5 and 13.5 dpc and intense expression was detected within the vascular smooth muscle layer in 16.5 dpc embryos. At later developmental time points, discrete areas of ACLP expression were detected in the mesenchymal cells in the dermal layer, developing skeletal structures, connective tissue, and in the umbilical ring and vessels. The predominance of ACLP immunoreactivity localized with collagen-rich regions including tendons and basement membranes. Overall, the developmental expression pattern is consistent with a regulatory or structural role in the abdominal wall, vasculature, and dermis.     

Calibration of ADHD Assessments Across Studies: A Meta-Analysis Tool

When analyzed separately, data from small studies provide only limited information with limited clinical generalizability, due to small sample size, differing assessments, and limited scope. In this methodological paper we outline a theoretical framework for performing meta-analysis of data obtained from disparate studies using disparate tests, based on calibration of the data from such studies and tests into a unified probability scale. We apply this method to combine the data from five studies examining the diagnostic abilities of different assessments of Attention Deficit/Hyperactivity Disorder (ADHD), including behavioral rating scales and EEG assessments. The studies enrolled a total of 111 subjects, 56 ADHD and 55 controls. Each individual study had a small sample focused on a specific age/gender group, for example 8 boys ages 6–10, and generally had insufficient power to detect statistically significant differences. No gender, or age comparisons were possible within any single study. However, when calibrated and combined, the data resulted in a clear separation between ADHD versus non-ADHD groups in males below the age of 16 (p < 0.001), males above the age of 16, (p = 0.015), females below the age of 16, (p = 0.0014), and females above the age of 16, (p = 0.0022).We conclude that if data from various studies using various tests are made comparable, the resulting combined sample size and the increased diversity of the combined sample lead to increased significance of the statistical tests and allow for cross-sectional comparisons, which are not possible within each individual study.     

Nuclear factor of activated T cells and serum response factor cooperatively regulate the activity of an alpha-actin intronic enhancer

Expression of alpha-actin in smooth muscle cells (SMCs) is regulated, in part, by an intronic serum response factor (SRF)-binding CArG element. We have identified a conserved nuclear factor of activated T cells (NFAT) binding site that overlaps this CArG box and tested the hypothesis that this site plays a previously unrecognized role in regulating alpha-actin expression. A reporter construct prepared using a 56-bp region of the mouse alpha-actin first intron containing SRF, NFAT, and AP-1 sites (SNAP) acted as an enhancer element in the context of a minimal thymidine kinase promoter. Basal reporter activity following expression in SMCs was robust and sensitive to the calcineurin-NFAT pathway inhibitors cyclosporin A and FK506. Mutating either the NFAT or SRF binding site essentially abolished reporter activity, suggesting that both NFAT and SRF binding are required. Basal activity in non-smooth muscle HEK293 cells was SRF-dependent but NFAT-independent and approximately 8-fold lower than that in SMCs. Activation of NFAT in HEK293 cells induced an approximately 4-fold increase in activity that was dependent on the integrity of both NFAT and SRF binding sites. NFATc3.SRF complex formation, demonstrated by co-immunoprecipitation, was facilitated by the presence of SNAP oligonucleotide. Inhibition of the calcineurin-NFAT pathway decreased alpha-actin expression in cultured SMCs, suggesting that the molecular interaction of NFAT and SRF at SNAP may be physiologically relevant. These data provide the first evidence that NFAT and SRF may interact to cooperatively regulate SMC-specific gene expression and support a role for NFAT in the phenotypic maintenance of smooth muscle.     

Post-fire recovery of acorn production by four oak species in southern ridge sandhill association in south-central Florida

We examined post-fire recovery of two components of acorn production (percentage of bearing ramets [stems] and number of acorns per bearing ramet) for four species of oaks in southern ridge sandhill vegetation in south-central peninsular Florida. Annual counts of acorns on two white oaks (Quercus chapmanii and Q. geminata) and two red oaks (Q. laevis and Q. myrtifolia) were conducted annually (except in 1991) on two 2.7-ha grids from 1969 to 1998. A prescribed burn was conducted on one of the grids in May 1993. Newly sprouted ramets of both white oaks produced acorns during the first year following the fire, whereas red oaks required 3 yr (Q. myrtifolia) or 4 yr (Q. laevis) to produce acorns. The difference in the timing of post-fire acorn production between the white and red oak species reflected the difference in the number of years from flower bud initiation to mature acorns in the two groups, with the additional year-long lag in Q. laevis probably attributable to the fact that it is typically a tree rather than a shrub species. The data suggested that percentage of bearing ramets in the smallest size class of the two white oak species was markedly lower in the burned than unburned grid in the first year of post-fire acorn production and higher in the fifth year, but these trends were not evident for the red oaks. Among all four species, differences between mean number of acorns in burned and unburned grids were significant in only two cases (the largest size class of both white oak species in the fifth year). There was no evidence of recruitment from acorns on the burned grid, possibly due to the rapid redevelopment of the shrub layer because of low mortality of the extensive clonal root systems. Rapid post-fire recovery of acorn production in xeric fire-prone habitats is presumably the result of selection to increase the probability of recovery and persistence following sufficiently intense fires that result in high oak mortality. The timing and magnitude of post-fire acorn production in sandhill and other xeric Florida associations has a potential impact on a wide variety of insects, birds, and mammals that feed on acorns, as well as on the species with which they interact.     

Akt participation in the Wnt signaling pathway through Dishevelled

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.     

The dephosphorylation of phosphoglucomutase by nucleophilic reagents.

The phosphoryl group on the serine residue at the active site of phosphoglucomutase is presumed to undergo nucleophilic attack by the monophosphate substrates glucose 1- and glucose 6-phosphate to form glucose 1,6-diphosphate. Fluoride, hydroxylamine, and several thiol compounds have now been shown to serve as effective nucleophiles toward the active phosphate and result in the dephosphorylation of phosphoglucomutase. The more extensively studied nucleophiles, cysteine, hydroxylamine, and fluoride, are effective at a concentration as low as 1 mM with a relative reactivity of 40, 2, and 1, respectively. The reaction proceeds as long as the catalytic activity of the enzyme is maintained. Inactivation of the enzyme abolishes dephosphorylation by all nucleophilic reagents thus far studied. The dephosphorylation reaction shows optimal activity of pH 6.5. The rate of dephosphorylation exhibits saturation kinetics. With fluoride the Km is 534 mM. Dephosphorylation by fluoride is stimulated by some but not all bivalent cations. Cu+ and Co2+ are the most effective. Cu2+ not only augments the reaction with fluoride but also facilitates a nucleophilic attack by water, in the absence of the halogen, to yield inorganic phosphate. No augmentation of the rate of dephosphorylation by bivalent cations can be elicited with either cysteine or hydroxylamine. The products of the fluoride reaction are phosphorofluoridate, a small but variable amount of inorganic phosphate, and a fully active dephosphoenzyme. By constrast, cysteine and hydroxylamine yield inorganic phosphate and a partially inactive enzyme. The dephosphorylation rate varies with temperature. Arrhenius plots for the fluoride reaction reveal two distinct slopes. The heat of activation between 5-37 degrees was found to be 10.2 Cal per mol. Between 0-5 degrees, however, it was considerably greater amounting to 24.3 Cal per mol.