Technical aspects of the Haematoxylin-Basic Fuchsin-picric acid (HBFP) stain applied to skeletal muscle

Synopsis The Haematoxylin-Basic Fuchsin-picric acid (HBFP) stain, a new non-enzymatic histochemical technique described previously to detect early myocardial ischemia, was applied to skeletal muscle. Several factors were found which have an important effect on HBFP positivity including ageing in room air of unstained tissue sections, and the precise timing of the differentiation step of this stain. Using carefully standardized techniques, repeatable staining was obtained and a high level of inter-observer consistency in the interpretation of staining results was achieved. Although the technical requirements of this new stain are rigorous, it offers promise and deserves further evaluation in the study of skeletal as well as cardiac muscle disorders. The histological advantages include vivid contrasts and the ability to use the stain on formalin-fixed paraffin-embedded muscle tissue.     

Purification and characterization of laccase II of Aspergillus nidulans

Sexual development in Aspergillus nidulans is a morphogenetic differentiation process triggered by internal and environmental signals. As a first step in analyzing the developmental pathway at the molecular level, laccase II (EC 1.10.3.2), which is specifically expressed in early stages of fruitbodies, was isolated. The enzyme was purified to apparent homogeneity from a mutant strain (SMS1) in which the sexual cycle dominates and the number of cleistothecia is increased tenfold. Laccase II was enriched 560-fold to a specific activity of 892 U (mg protein)^–1. The apparent molecular mass was determined to be 80 kDa under denaturing conditions and to be 100–120 kDa under native conditions. The internal peptide sequences gained from the protein will allow the isolation of the corresponding gene as a first step in determining the key regulators of sexual development. Received: 8 January 1998 / Accepted: 14 April 1998     

Separation of tonoplast and plasma membrane H+-ATPase from Zucchini hypocotyls by consecutive sucrose and glycerol gradient centrifugation

Summary In order to isolate tonoplast and plasma membrane vesicles involved in ATP-dependent proton transport we devised a preparative procedure with two consecutive centrifugations. Three fractions were obtained on a sucrose step gradient: light microsomes, heavy microsomes, and a mitochondria-rich fraction. The light and heavy microsomal fractions were each recentrifuged on an isopycnic glycerol density gradient. Recentrifugation of light microsomes resulted in two fractions with H⁺-ATPase activity, one equilibrating at a density less than 1.11 g/cm³ and one equilibrating at a density of about 1.17g/cm³. Comparison with marker enzyme activities suggests that the upper fraction was enriched in tonoplast, and the dense fraction with plasma membrane. In addition to marker enzyme content, H⁺ transport in the H⁺-ATPase-containing fractions was further characterized with respect to pH dependence, cation and anion dependence, and uncouplers and inhibitors. H⁺ transport in all fractions was strongly dependent on the presence of halides but no specific stimulation by potassium or any other monovalent cation was found. Of the anions tested, malate and fumarate preferentially stimulated H⁺ transport in the tonoplast-enriched fraction. It is suggested that a Ca²⁺/H⁺ antiporter is present in all fractions. Only H⁺-ATPase in the plasma membrane-enriched fractions was sensitive to nystatin, an uncoupler, and to orthovanadate, an inhibitor. The tonoplast fraction was more sensitive to nitrate than the plasma membrane-enriched fraction, and all fractions showed some sensitivity to high concentrations of oligomycin. Oligomycin sensitivity was not due to the presence of mitochondria.     

A plant lipid and the platelet-activating factor stimulate ATP-dependent H+ transport in isolated plant membrane vesicles

A plant lipid was isolated from zucchini (Cucurbita pepo L.) membranes and from soybean (Glycine max [L.] Merr) phospholipids by thinlayer chromatography and further purified by high-performance liquid chromatography. This plant lipid was chromatographically very similar to the platelet-activating factor, an ether phospho-lipid with hormone-like properties found in mammals. Both the plant lipid and the platelet-activating factor stimulated ATP-dependent H⁺ transport in isolated membrane vesicles from zucchini hypocotyls.Abbreviations HPLC high-performance liquid chromatography - PAF platelet-activating factor     

First Record of Stygimys (Mammalia, Multituberculata, Eucosmodontidae) from the Paleocene (Puercan) Part of the North Horn Formation, Utah, and a Review of the Genus

A single tooth referred to Stygimys kuszmauli recovered at the Gas Tank Hill locality from Puercan strata of the North Horn Formation represents the first record of Stygimys from Utah. S. kuszmauli now occurs in both proposed north–south late Puercan biogeographic provinces, which reduces statistical support for provinciality. A new species from Texas, Stygimys vastus, is described. Of the five species previously referred to Stygimys, S. kuszmauli, Stygimys camptorhiza, Stygimys jepseni, and Stygimys teilhardi are valid, but Stygimys cupressus is synonymous with S. kuszmauli. The type of S. jepseni is restricted to a single p4 as the m1 thought to be from the same individual represents another multituberculate genus. Five sites, including Gas Tank Hill that comprise the Gas Tank Hill Local Fauna are widely dispersed in slumped strata which may differ in age, are located in a mostly floodplain depositional setting, and are small surface collected samples. Therefore, biogeographic analyses that compare the Gas Tank Hill Local Fauna to large samples collected from channel fills using screenwash techniques should be employed with caution. Stygimys has a relatively broad geographic and geochronologic distribution, but it is usually rare in Late Cretaceous and Early Paleocene mammalian assemblages. The recent recovery of Stygimys after more than 60 years of collecting in the North Horn Formation suggests that further sampling in other non-marine basins in Western North America will eventually yield additional specimens of the genus.