Triple gene block protein interactions involved in movement of Barley stripe mosaic virus

Barley stripe mosaic virus (BSMV) encodes three movement proteins in an overlapping triple gene block (TGB), but little is known about the physical interactions of these proteins. We have characterized a ribonucleoprotein (RNP) complex consisting of the TGB1 protein and plus-sense BSMV RNAs from infected barley plants and have identified TGB1 complexes in planta and in vitro. Homologous TGB1 binding was disrupted by site-specific mutations in each of the first two N-terminal helicase motifs but not by mutations in two C-terminal helicase motifs. The TGB2 and TGB3 proteins were not detected in the RNP, but affinity chromatography and yeast two-hybrid experiments demonstrated that TGB1 binds to TGB3 and that TGB2 and TGB3 form heterologous interactions. These interactions required the TGB2 glycine 40 and the TGB3 isoleucine 108 residues, and BSMV mutants containing these amino acid substitution were unable to move from cell to cell. Infectivity experiments indicated that TGB1 separated on a different genomic RNA from TGB2 and TGB3 could function in limited cell-to-cell movement but that the rates of movement depended on the levels of expression of the proteins and the contexts in which they are expressed. Moreover, elevated expression of the wild-type TGB3 protein interfered with cell-to-cell movement but movement was not affected by the similar expression of a TGB3 mutant that fails to interact with TGB2. These experiments suggest that BSMV movement requires physical interactions of TGB2 and TGB3 and that substantial deviation from the TGB protein ratios expressed by the wild-type virus compromises movement.     

Laminin-332 is a substrate for hepsin, a protease associated with prostate cancer progression

Hepsin, a cell surface protease, is widely reported to be overexpressed in more than 90% of human prostate tumors. Hepsin expression correlates with tumor progression, making it a significant marker and target for prostate cancer. Recently, it was reported that in a prostate cancer mouse model, hepsin up-regulation in tumor tissue promotes progression and metastasis. The underlying mechanisms, however, remain largely uncharacterized. Hepsin transgenic mice displayed reduced laminin-332 (Ln-332) expression in prostate tumors. This is an intriguing cue, since proteolytic processing of extracellular matrix macromolecules, such as Ln-332, is believed to be involved in cancer progression, and Ln-332 expression is lost during human prostate cancer progression. In this study, we provide the first direct evidence that hepsin cleaves Ln-332. Cleavage is specific, since it is both inhibited in a dose-dependent manner by a hepsin inhibitor (Kunitz domain-1) and does not occur when catalytically inactive hepsin is used. By Western blotting and mass spectrometry, we determined that hepsin cleaves the beta3 chain of Ln-332. N-terminal sequencing identified the cleavage site at beta3 Arg(245), in a sequence context (SQLR(245) LQGSCFC) conserved among species and in remarkable agreement with reported consensus target sequences for hepsin activity. In vitro cell migration assays showed that hepsin-cleaved Ln-332 enhanced motility of DU145 prostate cancer cells, which was inhibited by Kunitz domain-1. Further, hepsin-overexpressing LNCaP prostate cancer cells also exhibited increased migration on Ln-332. Direct cleavage of Ln-332 may be one mechanism by which hepsin promotes prostate tumor progression and metastasis, possibly by up-regulating prostate cancer cell motility.     

Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease

Oxalates stimulate alterations in renal epithelial cells and thereby induce calcium oxalate (CaOx) stone formation. Bacillus subtilis YvrK gene encodes for oxalate decarboxylase (OxdC) which degrades oxalate to formate and CO₂. The present work is aimed to clone the oxdC gene in a mammalian expression vector pcDNA and transfect into Human Embryonic Kidney 293 (HEK293) cells and evaluate the oxdC expression, cell survival rate and oxalate degrading efficiency. The results indicate cell survival rate of HEK293/pcDNAOXDC cells pre-incubated with oxalate was enhanced by 28%. HEK293/pcDNAOXDC cells expressing OxdC treated with oxalate, significantly restored antioxidant activity, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) generation compared with HEK293/pcDNA. Apoptotic marker caspase 3 downregulation illustrates HEK293/pcDNAOXDC cells were able to survive under oxalate-mediated oxidative stress. The findings suggest HEK293 cells expressing oxdC capable of degrading oxalate protect cells from oxidative damage and thus serve as a therapeutic option for prevention of CaOx stone disease.

     

Morphological and growth altering effects of Cisplatin in C. albicans using fluorescence microscopy

Background

Extrapulmonary manifestations of tuberculosis have become increasingly important in the era of HIV/AIDS.

Case presentation

We describe a case of tuberculosis (TB) dactylitis in a patient with AIDS who originated from the Ivory Coast. The diagnosis was established by direct visualization of acid-fast bacilli on joint fluid and bone biopsy of the proximal phalanx. Imaging of the chest revealed multiple bilateral nodules. Confirmation of the diagnosis was made by isolation of Mycobacterium tuberculosis from sputum and bone cultures.

Conclusion

Tuberculosis should be considered in patients with unusual soft tissue or skeletal lesions, especially when an immunosuppressive condition is present. Ziehl-Neelsen staining and culture of tissue obtained via surgical biopsy offer the most direct approach to diagnosis.     

Interaction of BAG1 and Hsp70 mediates neuroprotectivity and increases chaperone activity

It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAGDeltaC) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAGDeltaC compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAGDeltaC was expressed exclusively in the cytosol. Furthermore, cells expressing BAGDeltaC were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.     

The effect of the cyclopropyl group on the conformation of chemotactic formyl tripeptides

Certain formyl peptides are powerful chemoattractants towards neutrophils. In this study, several formyl tripeptides were synthesized and used to investigate the effects of different amino acid residues in position 1 on their ability to stimulate neutrophil chemotaxis. Pig neutrophil chemotaxis towards the formyl tripeptide, HCO-Ac(3)C-Leu-Phe-OMe 1, where Ac(3)C represents 1-amino-1-cyclopropane carboxylic acid, was observed. Pig neutrophil chemotaxis towards a very similar formyl tripeptide, HCO-Aib-Leu-Phe-OMe 2, where Aib represents alpha-amino isobutyric acid, was not observed. Compared to the isopropyl group, it was shown that the cyclopropyl group induces a greater percentage of the E conformation about the formamide functionality in these peptides. For 1 and 2, the E isomer distributions in CDCl3 are 36 and 9%, respectively. Since a major difference between these two peptides is the Z/E isomeric distribution, one implication is that the peptide-receptor site interactions involving the E conformer are more effective than those of the Z conformer. No pig neutrophil chemotaxis towards the formyl tripeptides, HCO-Ala-Leu-Phe-OMe 3 and HCO-Gly-Leu-Phe-OMe 4 was observed. These formyl tripeptides exhibit a low percentage of the E isomer, similar to that of peptide 2.     

Synthesis,characterization and binding studies of chromium(III) complex containing an intercalating ligand with DNA

A chromium(III) complex [Cr(DPPZ)(2)Cl(2)](+), where DPPZ is a planar bidentate ligand with an extended aromatic system, has been found to bind strongly to CT DNA with an apparent binding constant of (1.8+/-0.5)x10(7) M(-1). The effects of [Cr(DPPZ)(2)Cl(2)](+) on the melting temperature and the viscosity of DNA clearly show that the chromium(III) complex interacts with DNA intercalatively. Competitive binding study shows that the enhancement in emission intensity of ethidium bromide (EthBr) in the presence of DNA was quenched by [Cr(DPPZ)(2)Cl(2)](+) indicating that the Cr(III) complex displaces EthBr from its binding site in DNA. The binding of this complex has been found to bring about B to Z conformational transition in CT DNA as well as poly(dG-dC).poly(dG-dC). Molecular modeling study also shows that binding energy of the complex with d(GC)(12) is much higher than Dickerson model and d(AT)(12). Modeling studies show that [Cr(DPPZ)(2)Cl(2)](+) brings about twist in the DNA base pairs as well as phosphate ester backbone resulting in conformational transition in DNA.     

Photooxidation of DNA by a cobalt(II) tridentate complex

[Co(bzimpy)(2)], where bzimpy is 2,6-bis(benzimidazol-2-yl)pyridine was synthesized and characterized by ESI-MS (electrospray ionization mass spectrometry), UV-visible and fluorescence spectra. Absorption titration and thermal denaturation experiments indicate that the complex binds to DNA with moderate strength. Viscosity measurement shows that the mode of binding could be surface binding. Fluorescence study shows that the fluorescence intensity of the complex decreases with increasing concentrations of DNA, which is due to the photoelectron transfer from guanine base to excited MLCT (metal to ligand charge transfer) state of the complex. Photoexcitation of the complex in the MLCT region in the presence of plasmid DNA has been found to give rise to nicking of DNA.     

Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs

The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms. Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied. Here we describe an alternative assay for determining the mRNA sites at which ribosomes or other factors are bound. This approach uses primer extension inhibition, or "toeprinting," to map the 3' boundaries of mRNA-associated complexes. This methodology, previously used to characterize initiation mechanisms in prokaryotic and eukaryotic systems, is used here to gain an understanding of two interesting translational regulatory phenomena in the fungi Neurospora crassa and Saccharomyces cerevisiae: (a) regulation of translation in response to arginine concentration by an evolutionarily conserved upstream open reading frame, and (b) atypical termination events that occur as a consequence of the presence of premature stop codons.     

Variable incidence angle fluorescence interference contrast microscopy for z-imaging single objects

Surface-generated structured illumination microscopies interrogate the position of fluorescently labeled objects near surfaces with nanometer resolution along the z axis. However, these techniques are either experimentally cumbersome or applicable to a limited set of experimental systems. We present a new type of surface-generated structured illumination fluorescence microscopy, variable incidence angle fluorescence interference contrast microscopy (VIA-FLIC), in which the fluorescent sample is assembled above a reflective Si surface and the incidence angle of excitation light is varied by placing annular photomasks with different radii in the aperture diaphragm plane of the microscope. The variation in incidence angle alters the interference pattern of excitation light, and hence the intensity of detected fluorescence. Quantitative VIA-FLIC is tested by using a set of fluorophore-containing supported membranes separated from the Si surface by SiO2 layers of variable thicknesses. The resulting fluorescence intensity versus incidence angle curves depends on the separation from the Si surface and when fit with an appropriate model yield precise SiO2 thicknesses that are accurate with respect to the known SiO2 thicknesses. Since only a simple modification to a standard epifluorescence microscope is required, VIA-FLIC offers a versatile method to produce z-reconstructions with high resolution for a wide range of biological systems.