Synthesis of N-acetyl-D-galactosamine and folic acid conjugated ribozymes

To evaluate potential improvement in tissue specific targeting and cellular uptake of therapeutic ribozymes, we have developed three new phosphoramidite reagents. These reagents can be used in automated solid-phase synthesis to produce oligonucleotide conjugates containing N-acetyl-D-galactosamine (targeting hepatocytes) and folic acid (targeting tumor). N-Acetyl-D-galactosamine was attached through a linker to both 2'-amino-2'-deoxyuridine and D-threoninol scaffolds, and these conjugates were converted to phosphoramidite building blocks. Incorporation of a D-threoninol-based monomer into ribozymes provided multiply labeled ribozyme conjugates. Attachment of the fully protected pteroic acid to the D-threoninol-6-aminocaproyl-L-glutamic acid construct afforded the folic acid conjugate, which was converted into the phosphoramidite and incorporated onto the 5'-end of the ribozyme.     

Heat- and cold-shock responses in <Emphasis Type="Italic">Fusarium graminearum</Emphasis> 3 acetyl- and 15 acetyl-deoxynivalenol chemotypes

Fusarium graminearum Schwabe is the primary cause of Fusarium head blight (FHB) in North America. Chemically distinct F. graminearum sub-populations can be identified based on the type or composition of deoxynivalenol (DON) mycotoxin derivatives, including 3-acetyl (3-ADON) and 15-acetyl (15-ADON). The evaluation of randomly selected 3-ADON and 15-ADON isolates, collected from spring wheat throughout Canada, was performed using thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ice-nucleation activity (INA), and heat and cold tolerance tests conducted within a temperature range of −70°C to 65°C. The results indicated that the 3-ADON sub-population, which is responsible for the highest disease severity and has rapidly displaced the 15-ADON sub-population, produces more DON and zearalenone (ZEA) than the 15-ADON sub-population when exposed to heat and cold. Following exposures (1 and 2 h) to extremely high or low temperatures, 3-ADON isolates exhibited faster mycelial growth than 15-ADON isolates. In addition, the warmest temperature at which INA activity occurred was in 3-ADON (−3.6°C) vs. 15-ADON (−5.1°C). Taken together, these features suggest that the newly emerging 3-ADON sub-population is more resilient than the resident 15-ADON sub-population. Overall, the differences between the two sub-populations could provide new insights into FHB epidemiology and if validated under field conditions, may provide important information for predicting future FHB epidemics.     

Chemiosmotic systems in bioenergetics: H+-cycles and Na+-cycles

The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H⁺ cycle concept. The circulation of H⁺ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H⁺ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes.Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H⁺. It was found that, in ceertain bacteria, as well as in the outer membrane of the animal cell, Na⁺ effectively substitutes for H⁺ as the coupling ion (the chemiosmotic Na⁺ cycle). A precedent is set when the Na⁺ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H⁺ and Na⁺ cycles coexist in one and the same membrane (bacteria) or in two diffeerent membranes of one and the same cell (animals). The sets of and generators as well as and consumers found in different types of biomembranes, are listed and discussed.     

Phosphorylation-dependent subcellular redistribution of small myosin light chain kinase

BackgroundMyosin light chain kinase (MLCK) is a Ca²⁺-calmodulin-dependent enzyme dedicated to phosphorylate and activate myosin II to provide force for various motile processes. In smooth muscle cells and many other cells, small MLCK (S-MLCK) is a major isoform. S-MLCK is an actomyosin-binding protein firmly attached to contractile machinery in smooth muscle cells. Still, it can leave this location and contribute to other cellular processes. However, molecular mechanisms for switching the S-MLCK subcellular localization have not been described.MethodsSite-directed mutagenesis and in vitro protein phosphorylation were used to study functional roles of discrete in-vivo phosphorylated residues within the S-MLCK actin-binding domain. In vitro co-sedimentation analysis was applied to study the interaction of recombinant S-MLCK actin-binding fragment with filamentous actin. Subcellular distribution of phosphomimicking S-MLCK mutants was studied by fluorescent microscopy and differential cell extraction.ResultsPhosphorylation of S-MLCK actin-binding domain at Ser25 and/or Thr56 by proline-directed protein kinases or phosphomimicking these posttranslational modifications alters S-MLCK binding to actin filaments both in vitro and in cells, and induces S-MLCK subcellular translocation with no effect on the enzyme catalytic properties.ConclusionsPhosphorylation of the amino terminal actin-binding domain of S-MLCK renders differential subcellular targeting of the enzyme and may, thereby, contribute to a variety of context-dependent responses of S-MLCK to cellular and tissue stimuli.General significanceS-MLCK physiological function can potentially be modulated via phosphorylation of its actin recognition domain, a regulation distinct from the catalytic and calmodulin regulatory domains.     

Climate Driven Life Histories: The Case of the Mediterranean Storm Petrel

Seabirds are affected by changes in the marine ecosystem. The influence of climatic factors on marine food webs can be reflected in long-term seabird population changes. We modelled the survival and recruitment of the Mediterranean storm petrel (Hydrobates pelagicus melitensis) using a 21-year mark-recapture dataset involving almost 5000 birds. We demonstrated a strong influence of prebreeding climatic conditions on recruitment age and of rainfall and breeding period conditions on juvenile survival. The results suggest that the juvenile survival rate of the Mediterranean subspecies may not be negatively affected by the predicted features of climate change, i.e., warmer summers and lower rainfall. Based on considerations of winter conditions in different parts of the Mediterranean, we were able to draw inferences about the wintering areas of the species for the first time.     

Structural and mechanical properties of TTR105-115 amyloid fibrils from compression experiments

Amyloid fibrils, originally associated with neurodegenerative diseases, are now recognized to have interesting mechanical properties. By using synchrotron x-ray diffraction at high pressure in a diamond anvil cell we determined the bulk modulus of TTR105-115 amyloid fibrils in water and in silicone oil to be 2.6 and 8.1 GPa, respectively. The compression characteristics of the fibrils are quite different in the two media, revealing the presence of cavities along the axis of the fibrils, but not between the β-sheets, which are separated by a dry interface as in a steric zipper motif. Our results emphasize the importance of peptide packing in determining the structural and mechanical properties of amyloid fibrils.     

Alteration of mitochondrial function and cell sensitization to death

Stimulation of cell death is a powerful instrument in the organism’s struggle with cancer. Apoptosis represents one mode of cell death. However, in a variety of tumor cells proapoptotic mechanisms are downregulated, or not properly activated, whereas antiapoptotic mechanisms are upregulated. Mitochondria are known as key players in the regulation of apoptotic pathways. Specifically, permeabilization of the mitochondrial outer membrane and subsequent release of proapoptotic proteins from the intermembrane space are viewed as decisive events in the initiation and/or execution of apoptosis. Disruption of mitochondrial functions by anticancer drugs, which induce oxidative stress, inhibit mitochondrial respiration, or uncouple oxidative phosphorylation, can sensitize mitochondria in these cells and facilitate outer membrane permeabilization.