Contaminant eluted from solid-phase plasmid affinity-purification protocol columns is not found using liquid-phase methods and can be prevented.

The preparation of high quality plasmid DNA is a necessary requirement for most molecular biology applications. We compared four different large plasmid preparation protocols, which were based on either a liquid-phase approach (Triton lysis) or purification of alkaline lysis bacterial extracts followed by supercoiled plasmid purification on affinity columns. Two host Escherichia coli strains, JM 109 and INValphaF', were used to grow the test plasmids for comparison of product plasmid DNA produced from the four different plasmid isolation methods. While the DNA grown in E. coli strain JM109, prepared by liquid-phase Triton lysis was appropriately restricted by 12 restriction enzymes, this was not the case for any of the JM109-grown DNA purified by any of the affinity column solid-phase approaches. In contrast to this, when the plasmid DNA was grown in E. coli strain INValphaF', most restriction enzymes cut DNA appropriately, irregardless of the plasmid preparation protocol used. It seems that an impurity commonly eluted with the DNA from all three of the solid-phase DNA columns had an equal effect on the above enzymes using the common host strain JM109, but not strain INValphaF'.     

Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus

The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.     

Novel Proteome Extraction Method Illustrates a Conserved Immunological Signature of MSI-H Colorectal Tumors

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Highlights

• •TOP: robust, bio-friendly FFPE proteome extraction method with less fixation bias.
• •Proteome of MSI-H colorectal cancer identifies immunobiology key elements.
• •MSI-H tumor displays an “INFg-STAT1 centric signature”.
• •Long-term IFNg induction In-vitro mimicks MSI-H signature.

     

Prostaglandin H synthase: protein synthesis-independent regulation in bovine aortic endothelial cells

The objective ofthe present study was to examine whether prostaglandin H synthase(PGHS) can be regulated by pathways independent of de novo synthesis ofPGHS. Incubation of bovine aortic endothelial cells (BAEC) for as shortas 5 min with NaF (40 mM) resulted in a 60% increase in PGHS activity.PGHS activity induced by NaF was unaffected by either 10 µMcycloheximide or 1 µM actinomycin D. Aspirin (25 µM) completelyinhibited resting PGHS activity, and NaF did not induce furtherstimulation. NS-398 (500 nM), a specific PGHS-2 inhibitor, wasineffective. Basic fibroblast growth factor (bFGF) induced asignificant increase in PGHS activity within 30 min and was insensitiveto cycloheximide. The levels of PGHS-1 and PGHS-2 proteins, as measuredby Western blots, were not affected by NaF or bFGF. The tyrosine kinaseinhibitor genistein attenuated PGHS activity that was induced by NaFand bFGF, whereas the tyrosine phosphatase inhibitor, sodiumorthovanadate, augmented these responses. The G protein activators5'-guanylyl imidodiphosphate and guanosine5'-O-(3-thiotriphosphate) inhibited both resting andNaF-induced PGHS activities. These results suggest that, in BAEC,PGHS-1 activity can be regulated by tyrosine kinase and/or Gproteins, independently of de novo protein synthesis.

     

Treatment with polyamines can prevent monosodium glutamate neurotoxicity in the rat retina

It has been previously shown that treatment of newborn rats with the polyamines putrescine, spermidine and spermine can rescue sympathetic neurons from naturally occurring cell death and from induced death after axotomy or immunosympathectomy. The present study demonstrates that polyamine treatment can also prevent the neurodegenerative effects in the retina and the loss of body weight caused by monosodium glutamate. The findings indicate that polyamine treatment may have a rather general beneficial effect on neuron survival.     

In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum

Glutamic acid producer Brevibacterium lactofermentum intact cells were used to demonstrate the feasibility of in vivo 15N NMR to follow nitrogen assimilation and amino acid production throughout the growth cycle. The induction of glutamic acid production by different growth conditions was studied. Intracellular and extracellular levels of free metabolites were estimated as function of oxygen supply and biotin concentration. 15N NMR enabled us to distinguish two phases during the fermentation. At the early stage of fermentation, glutamic acid was accumulated intracellularly independent of oxygen supply and no product was excreted. In the late growth phase, the permeability of the cells developed and L-glutamic acid was excreted. The effect of aeration and biotin concentration on cellular contents and excretion was also studied by 15N NMR. Glutamate, N-acetylglutamine, and glutamine were the main nitrogenous pools independent of cell culture conditions. Free ammonia was not accumulated intracellularly although glutamic acid fermentation can be characterized as the process of nitrogen assimilation and the uptake of ammonia is the key step. In conclusion, the application of in vivo 15N NMR spectroscopy unraveled various problems of nitrogen metabolism, in a rapid and nondestructive manner.