Measurement of cellular DNA mass by flow microfluorometry with use of a biological internal standard

Use of a biological standard (chicken erythrocytes) mixed with experimental cell populations allows control of all variables in flow microfluorometric determination of DNA content. These variables include both staining and instrument procedures. In addition, the use of a biological standard allows determination of cellular DNA mass in unperturbed cell populations. DNA mass measured by FMF technique correlates closely with values reported in the literature that used biochemical techniques.     

A comparison of two novel alcohol dehydrogenase enzymes (ADH1 and ADH2) from the extreme halophile Haloferax volcanii

Haloarchaeal alcohol dehydrogenases are exciting biocatalysts with potential industrial applications. In this study, two alcohol dehydrogenase enzymes from the extremely halophilic archaeon Haloferax volcanii (HvADH1 and HvADH2) were homologously expressed and subsequently purified by immobilized metal-affinity chromatography. The proteins appeared to copurify with endogenous alcohol dehydrogenases, and a double Δadh2 Δadh1 gene deletion strain was constructed to prevent this occurrence. Purified HvADH1 and HvADH2 were compared in terms of stability and enzymatic activity over a range of pH values, salt concentrations, and temperatures. Both enzymes were haloalkaliphilic and thermoactive for the oxidative reaction and catalyzed the reductive reaction at a slightly acidic pH. While the NAD⁺-dependent HvADH1 showed a preference for short-chain alcohols and was inherently unstable, HvADH2 exhibited dual cofactor specificity, accepted a broad range of substrates, and, with respect to HvADH1, was remarkably stable. Furthermore, HvADH2 exhibited tolerance to organic solvents. HvADH2 therefore displays much greater potential as an industrially useful biocatalyst than HvADH1.     

Assessing the uptake kinetics and internalization mechanisms of cell-penetrating peptides using a quenched fluorescence assay

Cell-penetrating peptides (CPPs) have shown great potency for cargo delivery both in vitro and in vivo. Different biologically relevant molecules need to be delivered into appropriate cellular compartments in order to be active, for instance certain drugs/molecules, e.g. antisense oligonucleotides, peptides, and cytotoxic agents require delivery into the cytoplasm. Assessing uptake mechanisms of CPPs can help to develop novel and more potent cellular delivery vectors, especially in cases when reaching a specific intracellular target requires involvement of a specific internalization pathway. Here we measure the overall uptake kinetics, with emphasis on cytoplasmic delivery, of three cell-penetrating peptides M918, TP10 and pVec using a quenched fluorescence assay. We show that both the uptake levels and kinetic constants depend on the endocytosis inhibitors used in the experiments. In addition, in some cases only the internalization rate is affected by the endocytosis inhibitors while the total uptake level is not and vice versa, which emphasizes importance of kinetic studies when assessing the uptake mechanisms of CPPs. Also, there seems to be a correlation between lower total cellular uptake and higher first-order rate constants. Furthermore, this may indicate simultaneous involvement of different endocytic pathways with different efficacies in the internalization process, as hypothesized but not shown earlier in an uptake kinetics assay.     

A new genus of African Acrometopini (Tettigoniidae: Phaneropterinae) based on morphology,chromosomes, acoustics,distribution, and molecular data,and the description of a new species

A new genus, Altihoratosphaga, is erected for species formerly assigned to Horatosphaga Schaum, 1853, and a new species is described. Four species are included in Altihoratosphaga: Altihoratosphaga nomima (Karsch, 1896), Altihoratosphaga montivaga ( Sjöstedt, 1909 ), Altihoratosphaga nou (Hemp, 2007) and Altihoratosphaga hanangensis sp. nov. All four species are restricted to Tanzanian localities, and, except for A. nomima, for which no ecological data are available, are confined to montane forest habitats. Data on ecology, acoustics, chromosomes, and molecular relationships are provided, as well as a key to Altihoratosphaga species. The present‐day distribution of Altihoratosphaga species suggests former migration events at times when wetter and colder climatic fluctuations favoured connections between montane forest communities, which today are isolated, enabling flightless taxa such as Altihoratosphaga and Monticolaria to spread. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 66–82.     

Cerivastatin enhances the cytotoxicity of 5-fluorouracil on chemosensitive and resistant colorectal cancer cell lines

Cerivastatin is one of the synthetic 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors used for the treatment and prevention of hypercholesterolaemia. The observation that patients receiving this drug had a lower incidence at cancer led to our interest in using it as a putative anticancer agent. In this study, we tested the cytotoxicity of cerivastatin on a panel of 5-fluorouracil (5FU) sensitive and resistant cell lines in vitro. Cerivastatin was cytotoxic to both 5FU sensitive and resistant cells. Cerivastatin significantly augmented the cytotoxic effect of 5FU on drug sensitive (6-22-fold) and resistant (229-310-fold) cell lines. Cerivastatin and 5FU acted synergistically. Cerivastatin inhibited nuclear factor kappaB DNA binding activity. The enhancing effect of cerivastatin on 5FU was partially mevalonate pathway independent. Cerivastatin may allow successful 5FU therapy in chemoresistant patients.     

Molecular mechanisms by which dietary isoflavones potentially prevent atherosclerosis

Dietary isoflavones are currently receiving much attention because of their potential role in preventing coronary artery disease and other chronic diseases. Accumulating evidence from cell culture and laboratory animal experiments indicates that isoflavones have the potential to prevent or delay atherogenesis. Suggested mechanisms of action include: a reduction in low-density lipoprotein (LDL) cholesterol and a potential reduction in the susceptibility of the LDL particle to oxidation; (2) an improvement in vascular reactivity; (3) an inhibition of pro-inflammatory cytokines, cell adhesion proteins and nitric oxide (NO) production; and (4) an inhibition of platelet aggregation. These mechanisms are consistent with the epidemiological evidence that a high consumption of isoflavone-rich soy products is associated with a reduced incidence of coronary artery disease. Biological effects of isoflavones are dependent on many factors, including dose consumed, duration of use, protein-binding affinity, and an individual's metabolism or intrinsic oestrogenic state. Further clinical studies are necessary to determine the potential health effects of isoflavones in specific population groups as we currently know little about age-related differences in exposure to these compounds and there are few guidelines on optimal dose for cardiovascular health benefits.     

Interaction of mithramycin with isolated GC and CG sites

We have studied the interaction of the GC-specific, minor groove-binding ligand, mithramycin, with cloned DNA inserts containing isolated GC and CG sites flanked by regions of (AT)_n and A_n · T_n using DNase I and hydroxyl radical footprinting. We find that mithramycin binds to GC better than CG and that AGCT is a better site than TGCA. Sites flanked by (AT)_n appear to be bound better than those surrounded by A_n · T_n. Although no footprints are produced at T₉GCA₉ and T₁₅CGA₁₅, DNase I cleavage is enhanced with the GC sites suggesting that there is some interaction with the ligand. Mithramycin also alters the DNase I cleavage of (GA)_n · (CT)_n.