Recombinant antibody libraries and selection technologies.

Active immunization has benefited human health perhaps more than any other biomedical advancement. Today, passive immunization is profoundly changing the practice of medicine by enabling antibody targeting of toxic, self, and other antigens not conducive to active immunization. Recombinant antibody libraries have contributed greatly to this progress and will continue to do so. The ability to construct and display a variety of antibody libraries, including naive, immune, semi-synthetic, and synthetic ones coupled with rapid screening and selection technologies, is in large measure responsible for the thousands of monoclonal antibody therapeutics in development.

Differential display in rat livers treated for 13 weeks with phenobarbital implicates a role for metabolic and oxidative stress in nongenotoxic carcinogenicity.

Hepatic enzyme inducers such as phenobarbital are often nongenotoxic rodent hepatocarcinogens. Currently, nongenotoxic hepatocarcinogens can only be definitively identified through costly and extensive long-term, repeat-dose studies (e.g., 2-year rodent carcinogenicity assays). Although liver tumors caused by these compounds are often not found to be relevant to human health, the mechanism(s) by which they cause carcinogenesis are not well understood. Toxicogenomic technologies represent a new approach to understanding the molecular bases of toxicological liabilities such asnongenotoxic carcinogenicity early in the drug discovery/development process. Microarrays have been used to identify mechanistic molecular markers of nongenotoxic rodent hepatocarcinogenesis in short-term, repeat-dose preclinical safety studies. However, the initial "noise" of early adaptive changes may confound mechanistic interpretation of transcription profiling data from short-term studies, and the molecular processes triggered by treatment with a xenobiotic agent are likely to change over the course of long-term treatment. Here, we describe the use of a differential display technology to understand the molecular mechanisms related to 13 weeks of dosing with the prototype rodent nongenotoxic hepatocarcinogen, phenobarbital. These findings implicate a continuing role for oxidative stress in nongenotoxic carcinogenicity.An Excel data file containing raw data is available in full at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. Click on the issue link for 33(1), then select this article. A download option appears at the bottom of this abstract. The file contains raw data for all gene changes detected by AFLP, including novel genes and genes of unknown function; sequences of detected genes; and animal body and liver weight ratios. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

High-throughput MALDI-TOF discovery of genomic sequence polymorphisms.

We describe a comparative sequencing strategy that is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of complete base-specific cleavage reactions of a target sequence. The target is converted to a DNA/RNA mosaic structure after PCR amplification using in vitro transcription. Cleavage with defined specificity is achieved by ribonucleases. The set of cleavage products is subjected to mass spectrometry without prior fractionation. The presented resequencing assay is particularly useful for single-nucleotide polymorphism (SNP) discovery. The combination of mass spectra from four complementary cleavage reactions detects approximately 98% of all possible homozygous and heterozygous SNPs in target sequences with a length of up to 500 bases. In general, both the identity and location of the sequence variation are determined. This was exemplified by the discovery of SNPs in the human gene coding for the cholesteryl ester transfer protein using a panel of 96 genomic DNAs.

RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis.

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.