AN AUTORADIOGRAPHIC AND HISTOCHEMICAL LOCALIZATION OF SULFATED POLYSACCHARIDES IN EUCHEUMA NUDUM (RHODOPHYTA)1

The predominant sulfated polysaccharide, ?-carrageenan, was localized in the middle lamella of epidermal, cortical and medullary cells of Eucheumanudum J. Agardh. Autoradiographic studies with ³⁵SO₄= indicated that the label was first incorporated in the inner wall and ultimately deposited in the middle lamella of all cells, and in an outer wall layer of the epidermal cells. There was no evidence for cytoplasmic incorporation of the label. The middle lamella stained with alcian blue, was γ-metachromatic with toluidina blue O and bound diaminobenzidine-osmium tetroxide. This region was also positive with periodic acid-Schiff's (PAS) ragent, possibly demonstrating cellulose and/or a nonsulfated precursor of ?-carrageenan. A proposed model for extracellular sulfation includes production and secretion of a nonsulfated polygalactan and sulfotransferase enzyme(s) by the golgi apparatus and endoplasmic reticulum, respectively. Free sulfate in the wall would be bound to the precursor polysaccharide, with much of the resulting carrageenan migrating to the middle lamella facilitating mutual cell growth.     

Über den »Cytotropismus« der Furchungszellen des Grasfrosches (Rana fusca)

Ohne ZusammenfassungNr. VIII der fortlaufenden, in verschiedene Zeitschriften vertheilten Serie von des Verfassers »Beiträgen zur Entwickelungsmechanik des Embryo«.     

Eine neue Weide aus dem Staate Washington

Ohne Zusammenfassung     

Increased Atmospheric SO2 Detected from Changes in Leaf Physiognomy across the Triassic–Jurassic Boundary Interval of East Greenland

The Triassic–Jurassic boundary (Tr–J; ∼201 Ma) is marked by a doubling in the concentration of atmospheric CO₂, rising temperatures, and ecosystem instability. This appears to have been driven by a major perturbation in the global carbon cycle due to massive volcanism in the Central Atlantic Magmatic Province. It is hypothesized that this volcanism also likely delivered sulphur dioxide (SO₂) to the atmosphere. The role that SO₂ may have played in leading to ecosystem instability at the time has not received much attention. To date, little direct evidence has been presented from the fossil record capable of implicating SO₂ as a cause of plant extinctions at this time. In order to address this, we performed a physiognomic leaf analysis on well-preserved fossil leaves, including Ginkgoales, bennettites, and conifers from nine plant beds that span the Tr–J boundary at Astartekløft, East Greenland. The physiognomic responses of fossil taxa were compared to the leaf size and shape variations observed in nearest living equivalent taxa exposed to simulated palaeoatmospheric treatments in controlled environment chambers. The modern taxa showed a statistically significant increase in leaf roundness when fumigated with SO₂. A similar increase in leaf roundness was also observed in the Tr–J fossil taxa immediately prior to a sudden decrease in their relative abundances at Astartekløft. This research reveals that increases in atmospheric SO₂ can likely be traced in the fossil record by analyzing physiognomic changes in fossil leaves. A pattern of relative abundance decline following increased leaf roundness for all six fossil taxa investigated supports the hypothesis that SO₂ had a significant role in Tr–J plant extinctions. This finding highlights that the role of SO₂ in plant biodiversity declines across other major geological boundaries coinciding with global scale volcanism should be further explored using leaf physiognomy.     

Comparison of Eleven Methods for Genomic DNA Extraction Suitable for Large-Scale Whole-Genome Genotyping and Long-Term DNA Banking Using Blood Samples

Over the recent years, next generation sequencing and microarray technologies have revolutionized scientific research with their applications to high-throughput analysis of biological systems. Isolation of high quantities of pure, intact, double stranded, highly concentrated, not contaminated genomic DNA is prerequisite for successful and reliable large scale genotyping analysis. High quantities of pure DNA are also required for the creation of DNA-banks. In the present study, eleven different DNA extraction procedures, including phenol-chloroform, silica and magnetic beads based extractions, were examined to ascertain their relative effectiveness for extracting DNA from ovine blood samples. The quality and quantity of the differentially extracted DNA was subsequently assessed by spectrophotometric measurements, Qubit measurements, real-time PCR amplifications and gel electrophoresis. Processing time, intensity of labor and cost for each method were also evaluated. Results revealed significant differences among the eleven procedures and only four of the methods yielded satisfactory outputs. These four methods, comprising three modified silica based commercial kits (Modified Blood, Modified Tissue, Modified Dx kits) and an in-house developed magnetic beads based protocol, were most appropriate for extracting high quality and quantity DNA suitable for large-scale microarray genotyping and also for long-term DNA storage as demonstrated by their successful application to 600 individuals.     

Distinct binding sites of Ala48-hirudin1-47 and Ala48-hirudin48-65 on alpha-thrombin

The interaction of alpha-thrombin with Ala48-hirudin, Ala48-hirudin1-47, and Ala48-hirudin48-65 was analyzed. Mutations at Pro48 were found to cause only slight changes in the kon (human: 3.1 +/- 0.3 x 10(8) M-1 s-1; bovine: 1.03 +/- 0.3 x 10(8) M-1 s-1) and koff (human: 0.4 +/- 0.2 x 10(-3) s-1; bovine: 2.9 +/- 0.4 x 10(-3) s-1) rate constants for the formation of the thrombin-hirudin complex. The amino-terminal fragment Ala48-hirudin1-47 containing the three disulfide bridges and the carboxyl-terminal fragment Ala48-hirudin48-65 were derived from the Ala48 mutant by proteolysis with endoproteinase Lys-C. These fragments inhibit bovine alpha-thrombin clotting activity with IC50 values of 0.6 and 4.9 microM, respectively (2.4 nM for r-hirudin). By mapping the interaction of Ala48-hirudin-derived fragments with bovine alpha-thrombin by limited proteolysis with trypsin and pancreatic elastase distinct binding sites for each fragment were determined. The carboxyl-terminal fragment was found to bind to the proposed anion-binding exosite in the region B62-74, whereas the amino-terminal fragment binds to a region around the elastase cleavage site at residues 150-151 of the alpha-thrombin B-chain.