Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides

Although the sequence specificity, biostability, and low toxicity of PMO (phosphorodiamidate morpholino oligomers) make them good antisense agents to study gene function, their limited ability to cross cell membranes limits their use in cell culture. In this paper we show that conjugation to arginine-rich peptides significantly enhanced the cellular uptake of PMO. The factors that affect the conjugate's cellular uptake and its antisense activity toward a targeted mRNA were investigated. Factors studied include the number of arginines in the peptide, the choice of cross-linker, the peptide conjugation position, the length of the PMO, and the cell culture conditions. Delivery of PMO to the cell nucleus and cytosol required conjugation rather than complexation of peptides to PMO. R(9)F(2)C was best suited to deliver a PMO to its target RNA resulting in the strongest antisense effect. By simply adding the R(9)F(2)C-PMO conjugate into the cell culture medium at low microM concentration, missplicing of pre-mRNA was corrected. This particular peptide-conjugated PMO was more effective than the PMO conjugated to the transmembrane transport peptides of HIV-1 Tat protein, Drosophila antennapedia protein, or to peptides with fewer arginines. Length of PMO did not affect a peptide's delivery efficacy, but all other factors were important. R(9)F(2)C peptide provided a simple and efficient delivery of PMO to a RNA target. Conjugation of peptide to PMO enhances the opportunities to evaluate gene functions in cell cultures.     

Serum equol, bone mineral density and biomechanical bone strength differ among four mouse strains

The extent of conversion of daidzein to its metabolite, equol, by intestinal microflora may be a critical step that determines if a diet rich in daidzein protects against the deterioration of bone after estrogen withdrawal. The objective was to determine the extent that daidzein is converted to equol. In addition, bone mineral content (BMC), bone mineral density (BMD) and strength of femurs and lumbar vertebrae (LV) in four mouse strains were measured. Mice were ovariectomized and fed control diet (AIN93G) with or without daidzein (200 mg daidzein/kg diet) for 3 weeks, after which serum, femurs and LV were collected. Serum daidzein and equol were elevated in all mice fed daidzein. Among mice fed daidzein, the CD-1 and Swiss–Webster (SW) mice had higher (P<.001) serum equol than C57BL/6 (C57) and C3H mice. Differences due to mouse strain were observed for all bone outcomes. C57 mice had lower femur BMC (P<.001), BMD (P<.001) and peak load at femur midpoint (P<.001) and neck (P<.001) than other mouse strains. C57 mice also had a lower femur midpoint yield load (P<.001) and resilience (P<.001) than C3H mice. C57 mice had a lower LV1–4 BMC (P<.001) and BMD (P<.001) compared with all mouse strains and peak load of LV3 was lower than CD-1 and SW mice. Differences in serum equol, BMD and bone strength properties should be considered when selecting a mouse strain for investigating whether dietary strategies that include isoflavones preserve bone tissue after ovariectomy.     

Major gaps in the distribution of protected areas for threatened and narrow range Afrotropical plants

We investigated the major patterns of plant rarity in sub-Saharan Africa, and looked for the most significant gaps in the reserve network of the region in terms of representing the distribution of threatened and geographically rare plants. Comparisons of the species ranges captured by the network of reserves were made against the proportion of species captured by randomly generated sets of areas and against a theoretical near minimum set of areas that represent all species once. At this scale of analysis, the network of large IUCN-coded reserves (the official ‘protected areas’) performs poorly against random and systematic selection procedures. Significant gaps in the IUCN-coded protected areas are in coastal Gabon/Cameroon, in the various tropical montane forest areas (Cameroon Highlands, Eastern Arc Mountains, Ethiopian Mountains), in lowland coastal eastern Africa, and in the South African Cape. Some of these gaps, for example in the Eastern Arc and eastern African coastal regions, are covered on the ground by a network of Forest Reserves under the management of national Forestry Authorities. The networks of Forest Reserves in Ghana, Nigeria, Cameroon, Uganda, Kenya, Zimbabwe, Zambia and Sierra Leone also fill reservation gaps for rare African plants in these countries. Upgrading the conservation status of some key Forest Reserves, which has been gradually happening for some decades, is proposed as an efficient way to enhance the protected area network of the Afrotropical region for the conservation of rare African plant species.     

Δ2-Troglitazone promotes cytostatic rather than pro-apoptotic effects in breast cancer cells cultured in high serum conditions

We have previously shown that Δ2-Troglitazone (Δ2-TGZ) displayed anticancer effects on breast cancer cell lines grown in low serum conditions (1% fetal calf serum (FCS)). The present study was performed in order to characterize the effects of Δ2-TGZ in high serum containing medium and to determine if starvation could influence the response of breast cancer cells to this compound, keeping in mind the potential interest for breast cancer therapy. We observed that in high serum conditions (10% FCS), a 48 h treatment with Δ2-TGZ induced a decrease in cell numbers in MDA-MB-231 and MCF-7 breast cancer cell lines. The IC₅₀ values were higher than in low serum conditions. Furthermore, in contrast to our previous results obtained in 1% FCS conditions, we observed that in 10% FCS-containing medium, MCF-7 cells were more sensitive to Δ2-TGZ than MDA-MB-231 cells. Δ2-TGZ also induced endoplasmic reticulum (ER) stress mainly in MDA-MB-231 cells. Besides, in high serum conditions, Δ2-TGZ induced a G₀/G₁ cell cycle arrest, an inhibition of BrdU incorporation and a reduced level of cyclin D1. We observed a limited cleavage of PARP and a limited proportion of cells in sub-G₁ phase. Thus, in high serum conditions, Δ2-TGZ displayed cytostatic effects rather than apoptosis as previously reported in 1% FCS-containing medium. Our results are in accordance with studies suggesting that serum starvation could potentiate the action of diverse anti-cancer agents.     

Comparison of the Effects of NADH- and NADPH-Perturbation Stresses on the Growth of <Emphasis Type="Italic">Escherichia coli</Emphasis>

To better understand how the reducing power of either NADH or NADPH affects cell growth, Escherichia coli strains expressing either NADH-dependent or NADPH-dependent azoreductase (EC 1.6.5.2), which mediates the reduction of an azo dye, were cultured in glucose minimal medium in the presence of 200 μM methyl red. Growth rates in NADH-perturbed, NADPH-perturbed, and control cells were 0.05, 0.12, and 0.13 h⁻¹, respectively. In addition, glucose consumption in NADH-perturbed cells was 10.8 g glucose/g cell, while that of control and NADPH-perturbed cells was very similar (3.6 vs 3.8 g glucose/g cell) during the perturbation phase. Therefore, NADH perturbation had a larger effect than NADPH on cellular growth. Susie Kim, Doo-Bum Moon contributed equally to this article.     

Amyloid fibrils as a nanoscaffold for enzyme immobilization

Amyloid fibrils are a misfolded state, formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them ideally suited as a readily functionalized nanoscaffold for enzyme immobilization and their strength, stability, and nanometer size are attractive features for exploitation in the creation of new bionanomaterials. We report successful functionalization of amyloid fibrils by conjugation to glucose oxidase (GOD) using glutaraldehyde. GOD retained activity upon attachment and successful cross‐linking was determined using electrophoresis, centrifugation, sucrose gradient centrifugation, and TEM. The resulting functionalized enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The antibacterial effect of the functionalized film was then tested on E. coli, the growth of which was inhibited, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. The incorporation of the GOD‐functionalized amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalized amyloid fibril scaffold. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cell-cycle-dependent variations in FTIR micro-spectra of single proliferating HeLa cells: principal component and artificial neural network analysis

We have previously reported spectral differences for cells at different stages of the eukaryotic cell division cycle. These differences are due to the drastic biochemical and morphological changes that occur as a consequence of cell proliferation. We correlate these changes in FTIR absorption and Raman spectra of individual cells with their biochemical age (or phase in the cell cycle), determined by immunohistochemical staining to detect the appearance (and subsequent disappearance) of cell-cycle-specific cyclins, and/or the occurrence of DNA synthesis. Once spectra were correlated with their cells' staining patterns, we used methods of multivariate statistics to analyze the changes in cellular spectra as a function of cell cycle phase.     

A mass spectrometry-based probe of equilibrium intermediates in protein-folding reactions

Described here is a mass spectrometry- and H/D exchange-based approach for the detection of equilibrium intermediate state(s) in protein-folding reactions. The approach utilizes the stability of unpurified proteins from rates of H/D exchange (SUPREX) technique to measure the m value (i.e., delta DeltaG/delta [denaturant] value) associated with the folding reaction of a protein. Such SUPREX m-value analyses can be made over a wide range of denaturant concentrations. Thus, the described approach is well-suited for the detection of high-energy intermediates that might be populated at low denaturant concentrations and hard to detect in conventional chemical denaturation experiments using spectroscopic probes. The approach is demonstrated on four known non-two-state folding proteins, including alpha-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin. The non-two-state folding behavior of each model protein system was detected by the described method. The cytochrome c, myoglobin, and IFABP systems each had high-energy intermediate states that were undetected in conventional optical spectroscopy-based studies and previously required other more specialized biophysical approaches (e.g., nuclear magnetic resonance spectroscopy-based methods and protease protection assays) for their detection. The SUPREX-based approach outlined here offers an attractive alternative to these other approaches, because it has the advantage of speed and the ability to analyze both purified and unpurified protein samples in either concentrated or dilute solution.     

Signal regulatory protein molecules are differentially expressed by CD8- dendritic cells

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpbeta1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8(-) cDC.