Azure a-Schiff, Alcian Blue, HIO4-Schiff, Naphthol Yellow S—A Sequential Staining Method for Paraffin Sections

Paraffin sections from tissue fixed 4-12 hr in 10% formalin containing 0.5% cetyl pyridinium chloride, and washed 2 hr, were stained as follows: (1) Hydrolyze in 5 N HCl at room temperature for 8.5-9 min, or use standard Feulgen hydrolysis at 60 C. (2) Stain in azure A-Schiff, 0.5% in bisulfite bleach (1 N HCl, 5; 10% Na₂S₂O₅, 5; and distilled water 90—parts by volume) for 10 min. (3) Place in bisulfite bleach 2 changes, 2 min each; wash in water, 1-2 min. (4) Stain in Alcian blue (0.1% in 0.01 2V HCl, pH 2.0) for 10 min. (5) Place in 0.01 N HCl for 2-3 min; wash in water for 1-2 min. (6) Oxidize in 0.5% HIO₄ for 5 min; wash in water, 1-2 min. (7) Stain in Schiff's leucofuchsiu, 10 min. (8) Treat with bisulfite bleach as in step 3; wash in running water, 10 min. (9) Stain in naphthol yellow S (0.01% in 1% acetic acid) for 1-2 min. (10) Place in 1% acetic acid for 2 min, dehydrate in tertiary butanol, clear and cover. Result: DNA is deep blue; acidic mucins are light blue; neutral polysaccharides, red to magenta; and proteins, yellow. Proper timing of the hydrolysis for the Feulgen reaction is the most critical step. Overhydrolysis results in green nuclei (staining by naphthol yellow S) whereas purplish nuclei are the results of insufficient hydrolysis.     

Occupancy of Mountain Plover and Burrowing Owl in Colorado

Abstract: Concern over the decline of grassland birds has spurred efforts to increase understanding of grassland bird-habitat relationships. Previous studies have suggested that black-tailed prairie dogs (Cynomys ludovicianus) provide important habitat for shortgrass prairie avifauna, such as mountain plover (Charadrius montanus) and western burrowing owl (Athene cunicularia hypugaea), although such studies are lacking in Colorado (USA). We used methods to estimate occupancy (ψ) of mountain plover and burrowing owl on prairie dog colonies and other shortgrass prairie habitats in eastern Colorado. Mountain plover occupancy was higher on prairie dog colonies (ψ = 0.50, 95% CI = 0.36–0.64) than on grassland (ψ = 0.07, 95% CI = 0.03–0.15) and dryland agriculture (ψ = 0.13, 95% CI = 0.07–0.23). Burrowing owl occupancy was higher on active prairie dog colonies (ψ = 0.80, 95% CI = 0.66–0.89) compared with inactive colonies (ψ = 0.23, 95% CI = 0.07–0.53), which in turn was much higher than on grassland (ψ = 0.01, 95% CI = 0.00–0.07) and dryland agriculture (ψ = 0.00, 95% CI ψ 0.00–0.00). Mountain plover occupancy also was positively correlated with increasing amounts of prairie dog colony in the landscape. Burrowing owl occupancy was negatively correlated with increasing amounts of prairie dog colony in the surrounding landscape. Our results suggest that actions to conserve mountain plovers and burrowing owls should incorporate land management to benefit prairie dogs. Because managing for specific colony attributes is difficult, alternative management that promotes heterogeneity may ensure that suitable habitat is available for the guild of grassland inhabitants.     

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.     

Historical and Ecological Factors in the Biogeography of Aposematic Neotropical Butterflies

SYNOPSIS. Quantitative analysis of low-level (mostly subspecific)geographic differentiation of aposematic color patterns in threegroups of forest butterflies (Heliconiini, Ithomiinae, and Troidini),in 1,550 half-degree quadrants throughout the Neotropics, indicatesfour principal species-endemicity regions, 44 principal subspecies-endemicitycenters, and 300 quadrants of subspecies hybridization. Theendemism centers are different in outline but nearly identicalin position for each of the three ecologically different groups.Basic data on present and past physical environments (climate,soils, geomorphology, and vegetation structures), when integratedinto maps, show 1) regions of favorable ecological conditionsfor forest organisms in the present, 2) regions of environmentalconformities and rapid transitions in the present, and 3) regionsof high probability for humid forest persistence during themajor landscape changes at the end of the last glacial period,13,000–20,000 yr ago. Species diversity (a local phenomenon)is readily related to favorable modern ecological conditionsand environmental microheterogeneity, promoted by unpredictablemild disturbance. Biogeographical patterns of evolution at thelevel of regional subspecies correlate very well with presumedpaleoecological conditions. The biogeographical data also supportan hypothesis of differentiation resulting from areas of rapidenvironmental change; most of the hybridization quadrants fallwithin transition zones. There is lower correlation betweencenters of endemism and zones of en vironmental conformity,but some individual cases of differentiation in the butterfliesmay be adequately explained by modern ecological factors. Speciespresently undergoing differentiation in isolated habitat islands(Amazonian savannas and high-altitude Andean forests and paramos)may offer the best opportunities for observation of evolutionaryprocesses in the present.     

Biomechanical and Morphometric Differences in Triticum aestivum Seedlings Differing in Rht Gene-Dosage

Biomechanical and morphometric comparisons among coleoptilesfrom wheat seedlings differing in Rht gene-dosage (Rht = 0,2, 4 doses) are presented in an effort to evaluate the influenceof Rht on the mechanics of soil penetration by this organ. Rhtis known to reduce seedling establishment compared to the wildtype. Data from 3–7-day-old seedlings indicate that Rhtreduces tissue elastic modulus E, increases the second momentof area I, and decreases the slenderness ratio (l/r) of coleoptiles.Rht-relatedchanges in E and I are such that the flexural stiffness of coleoptilesfrom Rht plants does not differ significantly from the wildtype-hence the growing coleoptiles of all three genotypes haveequivalent biomechanical capacity to penetrate the soil. Rhtreduction of coleoptile slenderness ratios confers a capacityto safely sustain higher axial compressive loads compared tocoleoptiles with equivalent flexural stiffness but higher ratios.However, wild type seedlings produce longer coleoptiles andlonger subcrown internodes than Rht seedlings. Longer coleoptilesdeliver the crown node closer to the top of the soil beforethe crown node extends beyond the lateral confinement of thecoleoptile. This reduces the potential for buckling of the subcrowninternode and leaves due to the compressive loading of soil.Rht affects a variety of mechanical features whose influenceis dependent upon the stage of seedling growth and the degreeof soil compaction. However, at equivalent depths of burialwhich exceed the maximum length of coleoptiles and moderatesoil compaction, Rht is biomechanically disadvantageous to seedlingestablishment. Wheat, germination, biomechanics, Rht-gene