Positive and Negative Selection

Cathepsins (i.e., cysteine proteinases) contribute to pathophysiological processes involving tissue damage, such as malignancies and chronic inflammation. Ideally, in order to understand them, their activity should be assayed in live cells. Rüttger et al. have developed a microplate assay for quantitative fluorometric determination of cathepsins B, H, K, and L activities in viable cells, without the need for cell fixation. They achieve this by trapping the diffusible product of enzymatic activity, 4-methoxy-β-naphthylamine, inside the cell by coupling it to 5-nitrosalicylaldehyde. This yields a yellow fluorescent, crystalline product. Using a combination of two partial selective factors—the substrates (peptidic derivatives of 4-methoxy-β-naphthylamide) and an inhibitor (E-64d)—Rüttger et al. then measured the activities of the above-mentioned cathepsins in a variety of cell types. Individual cathepsin activities were calculated as the difference between the assay including inhibitor and that lacking it. They validated the selectivity of their approach using macrophages isolated from cathepsin B^-/-, K^-/-, and L^-/- mice. -Page 469

Reactivating Inactive Xs

DNA methylation is an important method of gene silencing used in cellular processes as diverse as differentiation, genomic imprinting, X chromosome inactivation, and tumorigenic transformation. The demethylating drug 5-aza-2^′-deoxycytidine (5-aza-2dC) is often used to study DNA methylation. The standard protocol involves treating with the drug and then identifying activated genes. The problem is that cells in culture do not respond equally to 5-aza-2dC, as it is only incorporated into actively dividing cells. Therefore, treatment yields a heterogeneous population of cells with varying levels of demethylation. To circumvent this limitation, Vallender and Lahn have devised an approach that uses the reactivation of an X-inactivated GFP transgene as a reporter to identify only those cells that have undergone drug-induced demethylation. Flow cytometry is used to first isolate those primary cells whose GFP transgene resides on the randomly inactivated X chromosome (i.e., they don't express GFP). These cells are then treated with 5-aza-2dC, and those that do express GFP after treatment are subjected to microarray analysis to identify other genes (re)activated by demethylation. Combining this sorting technique with microarray analysis increased their list of differentially expressed genes by almost 2-fold. The authors suggest that their basic strategy can be exploited to study the biological effects of other chromatin-modifying drugs. -Page 461

A Bug's Life

The baculovirus expression vector system is widely used to overexpress recombinant proteins in Sf9 insect cells. It is popular because the insect cells allow posttranslational modifications, folding, and assembly of proteins—this cannot be accomplished in bacteria. Furthermore, high levels of expression are often achieved, and since Sf9 cells can be grown in suspension in serum-free media, large-scale experiments are possible. Ma et al. describe a direct and directional cloning strategy that generates recombinant baculo-viruses in only 1 week. They created an expression vector, pHBMBac-mid1.1, containing three unique Bsu36I sites (5^’-CCTNAGG-3^’); the wild-type baculovirus genome has none. The appropriate overhanging nucleotides are added to the primers when the target genes are amplified. The vectors are linearized by digestion with Bsu36I, ligated to the T4 DNA polymerasetreated PCR products containing the gene of interest (the authors used the DsRed fluorescent protein and mannanase), and the ligation mixtures are transfected directly into the Sf9 cells. One of the Bsu36I sites is embedded in the gfp gene, so complete digestion disrupts it and allows any background nonrecombinant baculoviruses to be easily visualized. The method is simple, convenient, quick, and cost-effective, and Ma et al. have made vectors containing His-tags, GST-tags, and FLAG®-tags to make it even more universally applicable. -Page 453

Don't aSCE Me Why

Researchers are increasingly becoming more interested in monitoring physiological processes at the single-cell level. This often requires the insertion of foreign molecules—dyes, drugs, DNA, and peptides—into individual cells. Single-cell electroporation (SCE) is a noninvasive technique that can accomplish this type of insertion by using electrokinetic force and diffusion across electric-field induced membrane pores at the end of a small electrode. Micropipet electrodes (MEs) are often utilized, but they pose a number of problems. First, they are difficult to manipulate; and second, SCE efficiency can be hampered by variable ME resistances, which may arise from inconsistencies in ME fabrication. In order to circumvent these issues, Bae and Butler have developed a new method of ME-based SCE with featuring two new aspects: (i) 40-nm precision feedback control of ME approach and (ii) a method to prescribe the applied membrane potential, V_m, a new electrical parameter for increased SCE efficiency. Their method is automated, hence called aSCE. Their improvements should ensure that aSCE will be used to enhance the design of electroporation protocols for rare and valuable reagents and cells. -Page 399

Epsin Silenced

A major hurdle in identifying which endocytotic pathway is utilized for the cellular uptake of any given ligand has been the development of an experimental strategy that suppresses one route exclusively. Approximately half of all known ligands are internalized through clathrin-coated pits; therefore, if one internalization pathway could be specifically shut down, this would be a good one to choose. Overexpression of dominant negative dynamin, a GTPase with an essential role in clathrin-mediated endocytosis, has been used to achieve this goal. However, there have been suggestions in the literature that this experimental strategy does not distinguish between caveolae- and clathrin-mediated uptake. Thus, Vanden Broeck and De Wolf have used RNAi to silence the expression of epsin, an essential accessory protein for clathrin-mediated uptake. Using human intestinal carcinoma cells, they generated a cell line (Caco-^2eps-) that constitutively and specifically suppresses epsin expression. They found that uptake of transferrin and EGF, both markers for clathrin-mediated endocytosis, was noticeably reduced (50%–75%) in these cells. -Page 475

It's All in the Family

The importance of epigenetic changes in disease states has become increasingly recognized; therefore studies exploring the role of chromatin in regulating gene expression have never been more relevant. However, restriction enzyme cleavage of intact nuclei, which is the classical method for determining chromatin accessibility, was designed to measure the accessibility of only single copy genes. Espinoza and Feeney have recently developed an assay to measure the chromatin accessibility of each individual gene within a highly homologous gene family. Their technique relies on a combination of ligation-mediated PCR and real-time PCR to yield data that is both sensitive and qualitative. In a proof-of-principle experiment, they checked the relative accessibility of the three members of the immunoglobulin V_HS107 family. Their starting material was 10⁶ freshly isolated mouse pro-B cells, in which this gene family is actively undergoing rearrangement. The results they obtained—that the V1 gene is more accessible than V11 or V13—agree with their known rearrangement frequencies: V1 rearranges five times more than V11, and 40 times more than V13. -Page 404