Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection.

Efforts have been made to improve the biological stability of phosphodiester (PO) oligonucleotides by the addition of various modifications to either the 3', 5' or both the 3' and 5' ends of an oligonucleotide. ISIS 1080, a phosphorothioate (PS) 21-mer oligonucleotide complementary to the internal AUG codon of UL13 mRNA in HSV-1, reduces the infectious yield of HSV-1 in HeLa cells to 9.0% +/- 11%. PO analogs of ISIS 1080 containing three PS linkages placed on the 3' (ISIS 1365), 5' (ISIS 1370), both the 3' and 5' (ISIS 1364) ends or with four linkages in the middle (ISIS 1400) demonstrated reduced antiviral efficacy compared to fully PS ISIS 1080. Thermal denaturation profiles demonstrated that these oligonucleotides hybridized to complementary DNA or RNA with equivalent binding affinities. All were able to support E. coli RNAse H cleavage of the HSV mRNA to which they were targeted. The stability of the congeners in cell culture medium containing 10% fetal calf serum (FCS), HeLa cytosolic extract, HeLa nuclear extract and in intact HeLa cells revealed that ISIS 1080 was most resistant to nucleolytic digestion through 48 hours. Partial PS oligonucleotides exhibited increased degradation compared to the fully thioated oligonucleotide by exonuclease activity in FCS and endonuclease activity in cell extracts or intact cells. Thus, the reduced efficacy of partial compared to fully PS oligonucleotides against HSV-1 in HeLa cells may result from increased degradation of the mixed PO/PS oligonucleotides.     

Relative spectral response as a function of sequential ligand binding

A unique method is presented for the determination of the critical number of ligands that must bind to a macromolecule to elicit a spectroscopic response. This method is based on analysis of ligand binding data. For example, four Ca2+ and two Mg2+ ions are necessary for mirroring the relative decrease in the intrinsic fluorescence of bovine prothrombin fragment 1. For application of the method, ligand loading and relative spectroscopic response data must be measured over a full range of concentrations.     

Dengue virus-specific human CD4+ T-lymphocyte responses in a recipient of an experimental live-attenuated dengue virus type 1 vaccine: bulk culture proliferation, clonal analysis, and precursor frequency determination.

We analyzed the CD4+ T-lymphocyte responses to dengue, West Nile, and yellow fever viruses 4 months after immunization of a volunteer with an experimental live-attenuated dengue virus type 1 vaccine (DEN-1 45AZ5). We examined bulk culture proliferation to noninfectious antigens, determined the precursor frequency of specific CD4+ T cells by limiting dilution, and established and analyzed CD4+ T-cell clones. Bulk culture proliferation was predominantly dengue virus type 1 specific with a lesser degree of cross-reactive responses to other dengue virus serotypes, West Nile virus, and yellow fever virus. Precursor frequency determination by limiting dilution in the presence of noninfectious dengue virus antigens revealed a frequency of antigen-reactive cells of 1 in 1,686 peripheral blood mononuclear cells (PBMC) for dengue virus type 1, 1 in 9,870 PBMC for dengue virus type 3, 1 in 14,053 PBMC for dengue virus type 2, and 1 in 17,690 PBMC for dengue virus type 4. Seventeen CD4+ T-cell clones were then established by using infectious dengue virus type 1 as antigen. Two patterns of dengue virus specificity were found in these clones. Thirteen clones were dengue virus type 1 specific, and four clones recognized both dengue virus types 1 and 3. Analysis of human leukocyte antigen (HLA) restriction revealed that five clones are HLA-DRw52 restricted, one clone is HLA-DP3 restricted, and one clone is HLA-DP4 restricted. These results indicate that in this individual, the CD4+ T-lymphocyte responses to immunization with live-attenuated dengue virus type 1 vaccine are predominantly serotype specific and suggest that a multivalent vaccine may be necessary to elicit strong serotype-cross-reactive CD4+ T-lymphocyte responses in such individuals.     

Iron supplementation moderates but does not cure the Belgrade anemia

Belgrade rats inherit microcytic, hypochromic anemia as an autosomalrecessive trait (gene symbol b). Erythrocytes and tissue are iron deficientin the face of elevated TIBC (total iron binding capacity) and percent ironsaturation; iron injections increased the number of erythrocytes but theirappearance remained abnormal. We have investigated iron supplements toimprove husbandry of b/b rats and to learn more about the underlying defectand its tissue distribution. Weekly IM (intramuscular) injections ofiron–dextran (Imferon at 30 mg kg) improved the anemia but did not alter thered cell morphology. Certain diets also improved the health of b/b rats whencompared to standard rat chows by the criteria of weight, survival toadulthood, hematology and reproduction. The critical nutritional factorturned out to be iron bioavailability, with ferrous iron added to the dietimproving the health of Belgrade rats without affecting the underlyingerythroid defect. Tissue iron measurements after dietary or parenteralsupplementation confirmed the iron deficient status of untreated b/b rats andestablished that dietary ferrous iron partially relieved this deficiency,with injections leading to greater amounts of tissue iron. Serum iron andTIBC were also found to be elevated in untreated b/b rats, with dietarysupplementation decreasing but not eliminating the elevation in TIBC. Thesestudies indicate that iron supplements can improve the health of b/b ratswithout altering the underlying defect and also suggest that the mutationcould alter iron uptake in the GI (gastrointestinal) tract.     

Recombination in Polymer:Fullerene Solar Cells with Open‐Circuit Voltages Approaching and Exceeding 1.0 V

Polymer:fullerene solar cells are demonstrated with power conversion efficiencies over 7% with blends of PBDTTPD and PC₆₁BM. These devices achieve open‐circuit voltages (V_oc) of 0.945 V and internal quantum efficiencies of 88%, making them an ideal candidate for the large bandgap junction in tandem solar cells. V_oc’s above 1.0 V are obtained when the polymer is blended with multiadduct fullerenes; however, the photocurrent and fill factor are greatly reduced. In PBDTTPD blends with multiadduct fullerene ICBA, fullerene emission is observed in the photoluminescence and electroluminescence spectra, indicating that excitons are recombining on ICBA. Voltage‐dependent, steady state and time‐resolved photoluminescence measurements indicate that energy transfer occurs from PBDTTPD to ICBA and that back hole transfer from ICBA to PBDTTPD is inefficient. By analyzing the absorption and emission spectra from fullerene and charge transfer excitons, we estimate a driving free energy of –0.14 ± 0.06 eV is required for efficient hole transfer. These results suggest that the driving force for hole transfer may be too small for efficient current generation in polymer:fullerene solar cells with V_oc values above 1.0 V and that non‐fullerene acceptor materials with large optical gaps (>1.7 eV) may be required to achieve both near unity internal quantum efficiencies and values of V_oc exceeding 1.0 V.     

Isolation of motoneuron cell bodies from spinal cord stored at -70 degrees C in ethylene glycol

Pretreatment of spinal cord with ethylene glycol permits long-term storage of the tissue at -70 degrees C prior to isolation and biochemical analysis of the cell bodies of spinal motoneurons. The method is useful for storing spinal tissue from laboratory animals, as well as from human post mortem specimens, where aliquots of tissue may then be used for motoneuron isolation over an indefinitely long period. In addition to inhibiting the loss of soluble proteins from the neurons during freezing and thawing, cryoprotection increases the yield and improves the appearance of the isolated cell bodies. The method should aid biochemical studies of many kinds of neuronal subpopulations isolated from small amounts of starting material.     

What Biomonitoring Can and Cannot Tell Us about Causality in Human Health and Ecological Risk Assessments

Biomonitoring can provide exposure and effects information on various stressors (chemical or biological) that can be useful for human health and ecological risk assessments. It has been applied over the years where harmful changes in human health or the environment were observed and which may have warranted more detailed investigation. Sometimes biomonitoring programs may have been useful in determining the significance and/or cause of these harmful observations. These data can help to infer, but not confirm, causality as exemplified in classical studies conducted in humans and wildlife. However, in most cases we note that additional work was needed to provide the information necessary to support or refute causality. Today modern technology provides the ability to measure a wide variety of parameters in environmental media, plants, animals, and humans. Finding a chemical in an environmental medium or biological tissue may be helpful in understanding potential exposure (and perhaps to begin estimating hazard) to humans and ecological receptors, but mere presence does not necessarily help to establish effects or assign causality. In this article we evaluate the strengths and weaknesses, in a risk assessment context, of the use of biomonitoring data to support a determination of causality.     

Desferal inhibits breast tumor growth and does not interfere with the tumoricidal activity of doxorubicin

Desferal is a clinically approved iron chelator used to treat iron overload. Doxorubicin is an anthracycline cancer chemotherapy drug used in the treatment of breast cancer. It can undergo redox cycling in the presence of iron to produce reactive oxygen species. The oxidant-generating activity of doxorubicin is thought to be responsible for the cardiotoxic side effects of the drug, but it is unclear whether it is also required for its anti-tumor activity. To test whether an iron-chelating antioxidant would interfere with the tumor-killing activity of doxorubicin, nude mice were transplanted with xenografts of human breast cancer MDA-MB 231 cells and then treated with doxorubicin and/or desferal. Not only did desferal not interfere with the anti-tumor activity of doxorubicin, it inhibited tumor growth on its own. In vitro studies confirmed that desferal inhibits breast tumor growth. However, it did not induce apoptosis, nor did it induce cell cycle arrest. Instead, desferal caused cytostasis, apparently through iron depletion. The cytostatic activity of desferal was partially ameliorated by pretreatment with iron-saturated transferrin, and transferrin receptor expression on breast cancer cells nearly doubled after exposure to desferal. In contrast to its effect on tumor cells, desferal did not inhibit growth of normal breast epithelial cells. The data indicate that the anti-tumor activity of doxorubicin is not dependent on iron-mediated ROS production. Furthermore, desferal may have utility as an adjunctive chemotherapy due to its ability to inhibit breast tumor growth and cardiotoxic side effects without compromising the tumor-killing activity of an anthracycline chemotherapy drug.