A review of selected triassic to Early Cretaceous ferns

After becoming nearly extinct during the Permian, the ferns began a slow recovery during the Triassic as the climate of the earth moderated. As a result, a considerable number and variety were present and widely distributed during the Jurassic and Early Cretaceous. However, with the rapid expansion of the angiosperms during the Late Cretaceous, the ferns once again became reduced in variety and greatly restricted in distribution. Some of the Mesozoic ferns are rather primitive and obviously are closely related descendants of Paleozoic taxa. Such ferns are assigned mostly to the Marattiaceae, Guaireaceae, Osmundaceae, and Gleicheniaceae. The majority of the Mesozoic ferns, however, are distinctive and appear to have originated during that era. These fossil ferns generally fit into modern orders and families such as the Matoniaceae or the Dipteridaceae. In some cases, it is difficult to clearly distinguish some of the Mesozoic ferns from living genera. A portion was presented as an invited paper to the symposium: Evolution of pteridophytes and gymnosperms at the XV International Botanical Congress, Yokohama, Japan (1993).     

The Late Triassic Schilderia Adamanica and Woodworthia Arizonica Trees of the Petrified Forest National Park, Arizona, Usa

Two fossil tree species, both with unusual characteristics, occur in the Upper Triassic of the Petrified Forest National Park, Arizona, USA and adjacent areas. The first, Schilderia adamanica , has a highly idiosyncratic secondary xylem structure which contains normal uniseriate and broad complex multiseriate 'herring-bone' rays. The trunk cross-section of the secondary xylem may be either of a normal pycnoxylic type with a central pith and rays radiating from that or it may consist of appressed 'xylem masses' with rays curving towards one another at their extremities. The second, Woodworthia arizonica , has narrow, horizontal vascular traces traversing the entire radial width of the secondary xylem. By analogy with extant tree species, these traces would have terminated on preventitious buds deeply embedded in the bark which, in the case of these fossil trunks, have failed to be silicified. Such buds have the capacity to develop into epicormic shoots when the crown foliage of the tree is damaged. A further specimen of W. arizonica is recorded for the first time from the Permian of southern Brazil. Reconstruction drawings of both trees are produced.     

Comparative evaluation of two purification methods of anti-CD19-c-myc-His6-Cys scFv

Different chromatographic methods have been used to purify bacterially expressed single chain antibodies in soluble or insoluble form. Here, we compared two methods for purification of anti-CD19-c-myc-His6-Cys scFv expressed in Escherichia coli as soluble protein. The protein-L-agarose purification method is a one step purification method that yielded significant amounts of pure protein compared to the two-step Ni-NTA-agarose plus Resource 15S purification method. However, the protein-L purification method exhibited an additional lower molecular weight protein contaminant. Based on results from in vitro gel digestion, mass spectrometry and database search results, we confirmed that the lower molecular weight protein contaminant, which could not be purified by Ni-NTA-agarose and 15S column method, is a degraded product of the full length scFv construct.     

Biotin carboxyl carrier protein Co-purifies as a contaminant in core-streptavidin preparations

We have successfully cloned and expressed core-streptavidin in Escherichia coli. Core-streptavidin was expressed in shaker flask culture as a soluble protein, isolated by periplasmic extraction, purified by immobilized metal affinity chromatography column, and analyzed for its size, thermal stability, and biotin-binding activity. In Western blots using streptavidin-horseradish peroxidase (HRP) as a probe, we identified a contaminant that co-purified with core-streptavidin, identified as biotin carboxyl carrier protein (BCCP). Although BCCP cannot be detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it appears as a prominent band in Western blot when probed with streptavidin peroxidase conjugate. Based on the results from in vitro gel digestion, mass spectrometry and Mascot database search results, we confirmed the presence of BCCP. It was found that BCCP can complex with core-streptavidin and can dissociate when heated above 80°C. BCCP could be successfully removed and recovered by using core-streptavidin immobilized magnetic beads under mild conditions. In addition, the enriched fractions of core-streptavidin oligotetramers were separated, which may be the by-products of BCCP binding to core-streptavidin in various ratios. Finally, enzyme linked immunosorbent assay results have shown that the amount of biotin-HRP binding to core-streptavidin was higher compared to commercially available streptavidin.     

X-ray analysis of Mycobacterium smegmatis Dps and a comparative study involving other Dps and Dps-like molecules

The structure of the DNA binding protein from starved cells from Mycobacterium smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M.smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches that they possess. The M.smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular 3-fold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M.smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces do not need to be between layers of protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.     

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T₀ plants with ASAL- lox-hpt-lox T-DNA and three single-copy T₀ plants with cre-bar T-DNA. Marker gene excisions were detected in T₁ hybrids through hygromycin sensitivity assay. Molecular analysis of T₁ plants exhibited 27.4% recombination efficiency. T₂ progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T₂ progeny plants. In planta bioassay of GLH and BPH performed on these T₂ progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.     

Mononuclear single helices of lanthanum(III) and europium(III). Metal dependent diastereomeric excess

Using the 1:2 condensate of benzildihydrazone and 2-acetylpyridine as a tetradentate N donor ligand L, LaL(NO₃)₃ (1) and EuL(NO₃)₃ (2), which are pale yellow in colour, are synthesized. While single crystals of 1 could not be obtained, 2 crystallises as a monodichloromethane solvate, 2·CH₂Cl₂ in the space group Cc with a = 11.7099(5) Å, b = 16.4872(5) Å, c = 17.9224(6) Å and β = 104.048(4)°. From the X-ray crystal structure, 2 is found to be a rare example of monohelical complex of Eu(III). Complex 1 is diamagnetic. The magnetic moment of 2 at room temperature is 3.32 BM. Comparing the FT-IR spectra of 1 and 2, it is concluded that 1 also is a mononuclear single helix. ¹H NMR reveals that both 1 and 2 are mixtures of two diastereomers. In the case of the La(III) complex (1), the diastereomeric excess is only 10% but in the Eu(III) complex 2 it is 80%. The occurrence of diastereomerism is explained by the chiralities of the helical motif and the type of pentakis chelates present in 1 and 2.     

Constitutive and UV-induced fibronectin degradation is a ubiquitination-dependent process controlled by beta-TrCP

Loss of fibronectin (FN) assembly in the extracellular matrix has long been recognized as a feature of cellular transformation. However, such assembly is regulated not only by FN synthesis but also by its post-translational modifications. The mechanism controlling FN protein stability has remained unclear so far. Recently it was demonstrated that FN matrix turnover occurs intracellularly at the lysosome following caveolin-1-dependent endocytosis. Although FN was reported to undergo ubiquitindependent degradation, the ubiquitin ligase responsible for FN ubiquitination is unknown. In this study, we have identified beta-TrCP as the ubiquitin ligase for lysosomal degradation of FN. We found two conserved beta-TrCP recognition motif (DSGVVYS and DSGSIVVS) in the primary amino acid sequence of human, mouse, and rat FN. Down-regulation of either beta-TrCP1 or beta-TrCP2 by small interference (siRNA) caused significant accumulation of FN. Immunolocalization studies showed intracellular accumulation of FN in beta-TrCP siRNA-treated cells without showing much alteration in its matrix association. We also observed that exposure of cells to UV irradiation effectively down-regulated FN following increased ubiquitination, which was significantly inhibited either by lysosomal inhibitor or by siRNA-mediated down-regulation of beta-TrCP. Taken together, constitutive FN degradation, as well as UV-induced degradation, is ubiquitination dependent and controlled by beta-TrCP.     

Protective role of S-adenosyl-L-methionine against hydrochloric acid stress in Saccharomyces cerevisiae

S-adenosyl-L-methionine (AdoMet, 1mM) protects the stationary phase cells of Saccharomyces cerevisiae against the killing effect of acid (10mM HCl, pH approximately 2). Both the acid and the acid plus AdoMet treatment for 2h increased the plasma membrane H(+)-ATPase activity; thereafter it decreased to the basal level. AdoMet partially recovered the intracellular pH (pH(in)) that dropped in presence of acid. AdoMet treatment facilitated acid induced phospholipid biosynthesis as well as membrane proliferation, which was reflected in the cellular lipid composition.