Mg-activated double-stranded DNA cleavage by [(bpy)2(OH2)Ru---O---Ru(H2O) (bpy)2]

The reaction of the title complex with DNA has been examined. Addition of [(bpy)₂(OH₂)RuORu(OH₂) (bpy)₂]⁴⁺ to DNA leads to the reduction of the complex to Ru(bpy)₂(OH₂)₂²⁺, as indicated by absorption spectroscopy and cyclic voltammetry. The reaction is accelerated by Mg²⁺. The combined evidence points to a mechanism where the oxo-bridged dimer is hydrolyzed to a monomeric Ru(III) complex that is capable of oxidizing DNA to effect strand scission. Gel electrophoresis demonstrates nicking of supercoiled /gfX174 DNA by [(bpy)₂(OH₂)RuORu(OH₂) (bpy)₂]⁴⁺, and double-stranded cleavage is observed in the presence of Mg²⁺. Linearization of the plasmid prior to treatment with the complex does not lead to further fragmentation, suggesting that supercoiling is required to realize double-stranded cleavage.     

Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.     

Modulating the Adhesion of Haematopoietic Stem Cells with Chemokines to Enhance Their Recruitment to the Ischaemically Injured Murine Kidney

Introduction

Renal disease affects over 500 million people worldwide and is set to increase as treatment options are predominately supportive. Evidence suggests that exogenous haematopoietic stem cells (HSCs) can be of benefit but due to the rarity and poor homing of these cells, benefits are either minor or transitory. Mechanisms governing HSC recruitment to injured renal microcirculation are poorly understood; therefore this study determined (i) the adhesion molecules responsible for HSC recruitment to the injured kidney, (ii) if cytokine HSC pre-treatment can enhance their homing and (iii) the molecular mechanisms accountable for any enhancement.

Methods

Adherent and free-flowing HSCs were determined in an intravital murine model of renal ischaemia-reperfusion injury. Some HSCs and animals were pre-treated prior to HSC infusion with function blocking antibodies, hyaluronidase or cytokines. Changes in surface expression and clustering of HSC adhesion molecules were determined using flow cytometry and confocal microscopy. HSC adhesion to endothelial counter-ligands (VCAM-1, hyaluronan) was determined using static adhesion assays in vitro.

Results

CD49d, CD44, VCAM-1 and hyaluronan governed HSC adhesion to the IR-injured kidney. Both KC and SDF-1α pre-treatment strategies significantly increased HSC adhesion within injured kidney, whilst SDF-1α also increased numbers continuing to circulate. SDF-1α and KC did not increase CD49d or CD44 expression but increased HSC adhesion to VCAM-1 and hyaluronan respectively. SDF-1α increased CD49d surface clustering, as well as HSC deformability.

Conclusion

Increasing HSC adhesive capacity for its endothelial counter-ligands, potentially through surface clustering, may explain their enhanced renal retention in vivo. Furthermore, increasing HSC deformability through SDF-1α treatment could explain the prolonged systemic circulation; the HSC can therefore continue to survey the damaged tissue instead of becoming entrapped within non-injured sites. Therefore manipulating these mechanisms of HSC recruitment outlined may improve the clinical outcome of cellular therapies for kidney disease.     

Combined Enzymatic and Physical Deinking Methodology for Efficient Eco-Friendly Recycling of Old Newsprint

Background

The development in the deinking process has made recycled fiber a major part of the raw material for pulp and paper industry. Enzymes have revolutionized the deinking process obtaining brightness levels surpassing conventional deinking processes. This study explores the deinking efficiencies of bacterial alkalophilic laccase (L) and xylanase (X) enzymes along with physical deinking methods of microwaving (MW) and sonication (S) for recycling of old newsprint (ONP).

Methods and Results

The operational parameters viz. enzyme dose, pH and treatment time for X and L deinking were optimized statistically using Response Surface Methodology. Laccase did not require any mediator supplementation for deinking. Deinking of ONP pulp with a combination of xylanase and laccase enzymes was investigated, and fiber surface composition and morphological changes were studied using X-ray diffraction, fourier transform infrared spectroscopy and scanning electron microscopy. Compared to the pulp deinked with xylanase (47.9%) or laccase (62.2%) individually, the percentage reduction of effective residual ink concentration (ERIC) was higher for the combined xylanase/laccase-deinked pulp (65.8%). An increase in brightness (21.6%), breaking length (16.5%), burst factor (4.2%) tear factor (6.9%), viscosity (13%) and cellulose crystallinity (10.3%) along with decrease in kappa number (22%) and chemical consumption (50%) were also observed. Surface appeared more fibrillar along with changes in surface functional groups. A combination of physical and enzymatic processes (S-MW-XL) for deinking further improved brightness (28.8%) and decreased ERIC (73.9%) substantially.

Conclusion

This is the first report on deinking of ONP with laccase without any mediator supplementation. XL pretreatment resulted in marked improvement in paper quality and a new sequence being reported for deinking (S-MW-XL) will contribute further in decreasing chemical consumption and making the process commercially feasible.     

Peanut Stripe Potyvirus Resistance in Peanut (Arachis Hypogaea L.) Plants Carrying Viral Coat Protein Gene Sequences

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.     

Liquid-based vs. conventional smears in fine needle aspiration of bone and soft tissue tumors

OBJECTIVE: To compare the accuracy of fine needle aspiration cytology of bone and soft tissue tumors utilizing ThinPrep (TP) (Cytyc Corporation, Boxborough, Massachusetts, U.S.A.) vs. conventional smears (CS). STUDY DESIGN: Fine needle aspiration cytology from bone and soft tissue tumors was processed and assessed for cellularity, nuclear and cytoplasmic preservation, cellular architecture and stromal background with both the TP liquid-based smear technique and conventional methods. RESULTS: An accurate diagnosis was made in 13% of TP cases as compared to 64% in CS cases. CONCLUSION: CS of fine needle aspiration sample is far superior to TP in diagnosing tumors of bone and soft tissues. Preservation of cytoplasmic features and cellular architecture was superior in conventionally prepared smears.     

Matrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge

An innovative method was developed for rapid sensitive detection and efficient structural characterization of lipopeptide biosurfactants by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry by using whole microbial cells and crude culture filtrates as targets in combination with surface tension measurements. This was done for a bacterial strain that was isolated from petroleum sludge and efficiently produces biosurfactants. This organism was identified by using biochemical, physiological, and genetic parameters as a Bacillus subtilis strain, designated B. subtilis C-1. This assignment was supported by a mass spectrometric investigation of the secondary metabolite spectrum determined by whole-cell MALDI-TOF mass spectrometry, which revealed three lipopeptide complexes, the surfactins, the iturins, and the fengycins, which are well-known biosurfactants produced by B. subtilis strains. These compounds were structurally characterized by in situ structure analysis by using postsource decay MALDI-TOF mass spectrometry. The isoforms were separated by miniaturized high-resolution reversed-phase high-performance liquid chromatography for mass spectrometric characterization. Iturin compounds which contain unusual fatty acid components were detected.     

Cell surface accumulation of a truncated transmembrane prion protein in Gerstmann-Straussler-Scheinker disease P102L

A familial prion disorder with a proline to leucine substitution at residue 102 of the prion protein (PrP(102L)) is typically associated with protease-resistant PrP fragments (PrP(Sc)) in the brain parenchyma that are infectious to recipient animals. When modeled in transgenic mice, a fatal neurodegenerative disease develops, but, unlike the human counterpart, PrP(Sc) is lacking and transmission to recipient animals is questionable. Alternate mice expressing a single copy of PrP(102L) (mouse PrP(101L)) do not develop spontaneous disease, but show dramatic susceptibility to PrP(Sc) isolates from different species. To understand these discrepant results, we studied the biogenesis of human PrP(102L) in a cell model. Here, we report that cells expressing PrP(102L) show decreased expression of the normal 18-kDa fragment on the plasma membrane. Instead, a 20-kDa fragment, probably derived from transmembrane PrP ((Ctm)PrP), accumulates on the cell surface. Because the 20-kDa fragment includes an amyloidogenic region of PrP that is disrupted in the 18-kDa form, increased surface expression of 20-kDa fragment may enhance the susceptibility of these cells to PrP(Sc) infection by providing an optimal substrate, or by amplifying the neurotoxic signal of PrP(Sc). Thus, altered susceptibility of PrP(101L) mice to exogenous PrP(Sc) may be mediated by the 20-kDa (Ctm)PrP fragment, rather than PrP(102L) per se.     

Characterization of gibberellin producing strains of Fusarium moniliforme based on DNA polymorphism

The gibberellins are one of the major groups of growth promoting hormones and are secondary metabolites of the fungus Fusarium moniliforme (Perfect stage: Gibberella fujikuroi). Sixteen strains of Fusarium from different geographical regions and different hosts were analysed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). Range of gibberellin production varied between 28.9 to 600.0 mg g^-1 dry weight of mycelium in different strains of Fusarium. RAPD analysis showed completely different pattern between high, moderate and low producing strains. High producers formed nearly identical RAPD patterns, whereas the low and moderate producers gave heterologous amplification patterns. Since Fusarium pallidoroseum was in another group, it was possible to distinguish between different species of the genus Fusarium by RAPD. These investigations may find an application in the diagnosis of unknown Fusarium species and in distinguishing isolates of Gibberella fujikuroi within the section of Liseola. This revised version was published online in June 2006 with corrections to the Cover Date.