A Simple Approach to Assign Disulfide Connectivity Using Extracted Ion Chromatograms of Electron Transfer Dissociation Spectra

Increasing interest in production of protein-based pharmaceuticals (biotherapeutics) is accompanied by an increased need for verification of protein folding and correct disulfide bonding. Recombinant protein expression may produce aberrant disulfide bonds and could result in safety concerns or decreased efficacy. Thus, the thorough analysis of disulfide bonding is a necessity for protein therapeutics. The use of ETD facilitates this analysis because disulfide bonds are preferentially cleaved when subjected to ETD. Here, we make use of this well-characterized reaction to assign disulfide bonding networks by coupling the use of extracted ion chromatograms (XICs) of cysteine-containing peptides with ETD analysis to produce an efficient assignment approach for disulfide bonding. This method can be used to assign a disulfide pattern in a de novo fashion, to detect disulfide shuffling, and to provide information on heterogeneity, when more than one disulfide bonding pattern is present. The method was applied for assigning the disulfide-bonding network of a recombinant monomer of the HIV envelope protein gp120. It was found that one region of the protein, the V1/V2 loops, had significant heterogeneity in the disulfide bonds

Generation of an integrated transcription map of the BRCA2 region on chromosome 13q12-q13

An integrated approach involving physical mapping, identifcation of transcribed sequences, and computational analysis of genomic sequence was used to generate a detailed transcription map of the 1.0-Mb region containing the breast cancer susceptibility locus BRCA2 on chromosome 13q12–q13. This region is included in the genetic interval bounded by D13S1444 and D13S310. Retrieved sequences from exon amplif-cation or hybrid selection procedures were grouped into physical intervals and subsequently grouped into transcription units by clone overlap. Overlap was established by direct hybridization, cDNA library screening, PCR cDNA linking (island hopping), and/ or sequence alignment. Extensive genomic sequencing was performed in an effort to understand transcription unit organization. In total, approximately 500 kb of genomic sequence was completed. The transcription units were further characterized by hybridization to RNA from a series of human tissues. Evidence for seven genes, two putative pseudogenes, and nine additional putative transcription units was obtained. One of the transcription units was recently identifed as BRCA2 but all others are novel genes of unknown function as only limited alignment to sequences in public databases was observed. One large gene with a transcript size of 10.7 kb showed signifcant similarity to a gene predicted by the Caenorhabditis elegans genome and the Saccharomyces cerevisiae genome sequencing efforts, while another contained a motif sequence similar to the human 2*, 3* cyclic nucleotide 3* phosphodies-terase gene. Several retrieved transcribed sequences were not aligned into transcription units because no corresponding cDNAs were obtained when screening libraries or because of a lack of defnitive evidence for splicing signals or putative coding sequence based on computational analysis. However, the presence of additional genes in the BRCA2 interval is suggested as groups of putative exons and hybrid selected clones that were transcribed in consistent orientations could be localized to common physical intervals. q 1996 Academic Press, Inc

Sedimentation Tanks for Treating Rainwater: CFD Simulations and PIV Experiments

The removal of solids is the most important step when treating rainwater. The article evaluates two designs of sedimentation tanks that can be used for the continuous separation of fine particles from water: OS—standard sedimentation tanks, and OW—swirl sedimentation tanks. The tanks were studied by conducting computational fluid dynamics (CFD) modeling and particle image velocimetry (PIV) experiments. The settling process in sedimentation tank was carried out at varying operating flow rates. A tank with a modified structure was used for the tests, where water was supplied by a nozzle placed at an angle. This solution made it possible to obtain a rotational flow that transported the suspended particles towards its wall, where downward axial velocity resulted in the settling of particles. Based on the research, it was observed that the flow patterns showed inward flow at the bottom of the tank and an upward flow and the lifting of the settled particles near the hatch at the bottom. The presented experimental measurements provided detailed insight into flow patterns, and valuable calibration and verification data for further CFD modeling. Traditional PIV techniques are useful in the case of standard design, whereas CFD is invaluable for supporting this work and for investigating the design of novel sedimentation tanks

Secretion of prostatic specific antigen, proliferative activity and androgen response in epithelial–stromal co-cultures from human prostate carcinoma

We investigate the proliferative activity, prostatic specific antigen (PSA) secretion, morphology and androgen response of human prostate tumour epithelial cells co-cultured with stromal cells in a bicameral system. Stromal and epithelial cells were isolated from prostate adenocarcinoma by enzyme digestion and cultured in defined media. Immunocytochemistry for prostate carcinoma tumour antigen (PCTA-1) was performed for culture purity evaluation. Also, the morphology of the epithelial cells in co-culture was evaluated by electron microscopy. PSA was determined by microparticle enzyme immunoassay (MEIA) automatized protocol and the proliferation was evaluated by a commercial spectrophotometric kit, based on formazan salt formation. Both cell cultures showed more than 90% of purity. The epithelial cell co-cultures showed marked membrane processes and cell interdigitations. The proliferative activity of the epithelial cells was increased in presence of stromal cells. Also, PSA secretion was significantly increased and maintained for at least 14 days, whereas the androgen response for PSA secretion was evidenced only in co-culture condition. Primary co-cultures of epithelial and stromal cells from human prostate carcinoma are able to maintain, for a prolonged time, proliferative and secretory properties as well hormone response, and represent a valuable tool for cellular and molecular studies on prostate cancer