Relationship between human IgG structure and retention time in hydroxyapatite chromatography with sodium-phosphate gradient elution.

The aim of this study was to classify the retention time of recombinant human monoclonal antibodies (rhMAbs) in hydroxyapatite chromatography with sodium-phosphate gradient elution according to their physicochemical properties. We analyzed 37 rhMAbs and found that (i) the structures of both the constant and variable regions affected retention time, (ii) the number of basic amino acid residues in the variable region, particularly in the heavy chain, correlated well with retention time, and (iii) this correlation was more pronounced (e.g. r(2)=0.87 for 18 kappa IgG(1) rhMAbs) when the surface accessibility of those residues are taken into account. These findings provide a useful guide for investigators and purification-process developers working with monoclonal antibodies.

Relationship between human IgG structure and retention time in hydroxyapatite chromatography with sodium chloride gradient elution.

Accurate prediction of the elution tendency of monoclonal antibodies in column chromatography would be beneficial for the efficient setup of purification procedures. Hydroxyapatite chromatography experiments using 37 recombinant human monoclonal antibodies were performed by sodium chloride gradient elution with 5 mM sodium phosphate to correlate the retention times with antibody structures (subclass and light-chain isotypes). The contribution of metal affinity interactions in the interaction of antibodies with hydroxyapatite was investigated by (i) eliminating 5 mM sodium phosphate in buffers, (ii) comparing sodium chloride versus sodium phosphate gradient elutions, and (iii) using IgG(4) antibodies with a leucine-->glutamate mutation. By using antibodies of different subclasses but with identical Fab regions, the elution behavior in sodium chloride elution could be classified by subclass and type of light chain. It is considered that the retention of monoclonal antibodies to hydroxyapatite is affected by the cooperation of phosphoryl cation exchange and metal affinity interactions. The contribution of the metal affinity interactions is greater in the sodium chloride gradient elution method than in the sodium phosphate gradient elution method.

TCP: a tool for designing chimera proteins based on the tertiary structure information.

BACKGROUND: Chimera proteins are widely used for the analysis of the protein-protein interaction region. One of the major issues is the epitope analysis of the monoclonal antibody. In the analysis, a continuous portion of an antigen is sequentially substituted into a different sequence. This method works well for an antibody recognizing a linear epitope, but not for that recognizing a discontinuous epitope. Although the designing the chimera proteins based on the tertiary structure information is required in such situations, there is no appropriate tool so far. RESULTS: In light of the problem, we developed a tool named TCP (standing for a Tool for designing Chimera Proteins), which extracts some sets of mutually orthogonal cutting surfaces for designing chimera proteins using a genetic algorithm. TCP can also incorporate and consider the solvent accessible surface area information calculated by a DSSP program. The test results of our method indicate that the TCP is robust and applicable to various shapes of proteins. CONCLUSION: We developed TCP, a tool for designing chimera proteins based on the tertiary structure information. TCP is robust and possesses several favourable features, and we believe it is a useful tool for designing chimera proteins. TCP is freely available as an additional file of this manuscript for academic and non-profit organization.

Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis.

The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.

A hidden Markov model-based approach for identifying timing differences in gene expression under different experimental factors.

MOTIVATION: Time series experiments of cDNA microarrays have been commonly used in various biological studies and conducted under a lot of experimental factors. A popular approach of time series microarray analysis is to compare one gene with another in their expression profiles, and clustering expression sequences is a typical example. On the other hand, a practically important issue in gene expression is to identify the general timing difference that is caused by experimental factors. This type of difference can be extracted by comparing a set of time series expression profiles under a factor with those under another factor, and so it would be difficult to tackle this issue by using only a current approach for time series microarray analysis. RESULTS: We have developed a systematic method to capture the timing difference in gene expression under different experimental factors, based on hidden Markov models. Our model outputs a real-valued vector at each state and has a unique state transition diagram. The parameters of our model are trained from a given set of pairwise (generally multiplewise) expression sequences. We evaluated our model using synthetic as well as real microarray datasets. The results of our experiment indicate that our method worked favourably to identify the timing ordering under different experimental factors, such as that gene expression under heat shock tended to start earlier than that under oxidative stress. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

PURE: a PubMed article recommendation system based on content-based filtering.

We have developed a PubMed article recommendation system, PURE, which is based on content-based filtering. PURE has a web interface by which users can add/delete their preferred articles. Once articles are registered, PURE then performs model-based clustering of the preferred articles and recommends the highly-rated articles by the prediction using the trained model. PURE updates the PubMed articles and reports the recommendation by email on daily-base. This system will be helpful for biologists to reduce the time required for gathering information from PubMed. PURE is downloadable under GPL license, via www.bic.kyoto-u.ac.jp/pathway/mami/out/PURE.tar.gz.

A novel transgenic chimaeric mouse system for the rapid functional evaluation of genes encoding secreted proteins.

A major challenge of the post-genomic era is the functional characterization of anonymous open reading frames (ORFs) identified by the Human Genome Project. In this context, there is a strong requirement for the development of technologies that enhance our ability to analyze gene functions at the level of the whole organism. Here, we describe a rapid and efficient procedure to generate transgenic chimaeric mice that continuously secrete a foreign protein into the systemic circulation. The transgene units were inserted into the genomic site adjacent to the endogenous immunoglobulin (Ig) kappa locus by homologous recombination, using a modified mouse embryonic stem (ES) cell line that exhibits a high frequency of homologous recombination at the Igkappa region. The resultant ES clones were injected into embryos derived from a B-cell-deficient host strain, thus producing chimaerism-independent, B-cell-specific transgene expression. This feature of the system eliminates the time-consuming breeding typically implemented in standard transgenic strategies and allows for evaluating the effect of ectopic transgene expression directly in the resulting chimaeric mice. To demonstrate the utility of this system we showed high-level protein expression in the sera and severe phenotypes in human EPO (hEPO) and murine thrombopoietin (mTPO) transgenic chimaeras.

PSE: a tool for browsing a large amount of MEDLINE/PubMed abstracts with gene names and common words as the keywords.

BACKGROUND: MEDLINE/PubMed (hereinafter called PubMed) is one of the most important literature databases for the biological and medical sciences, but it is impossible to read all related records due to the sheer size of the repository. We usually have to repeatedly enter keywords in a trial-and-error manner to extract useful records. Software which can reduce such a laborious task is therefore required. RESULTS: We developed a web-based software, the PubMed Sentence Extractor (PSE), which parses large number of PubMed abstracts, extracts and displays the co-occurrence sentences of gene names and other keywords, and some information from EntrezGene records. The result links to whole abstracts and other resources such as the Online Mendelian Inheritance in Men and Reference Sequence. While PSE executes at the sentence-level when evaluating the existence of keywords, the popular PubMed operates at the record-level. Therefore, the relationship between the two keywords, a gene name and a common word, is more accurately captured by PSE than PubMed. In addition, PSE shows the list of keywords and considers the synonyms and variations on gene names. Through these functions, PSE would reduce the task of searching through records for gene information. CONCLUSION: We developed PSE in order to extract useful records efficiently from PubMed. This system has four advantages over a simple PubMed search; the reduction in the amount of collected literatures, the showing of keyword lists, the consideration for synonyms and variations on gene names, and the links to external databases. We believe PSE is helpful in collecting necessary literatures efficiently in order to find research targets. PSE is freely available under the GPL licence as additional files to this manuscript.

Mutant FGF-23 responsible for autosomal dominant hypophosphatemic rickets is resistant to proteolytic cleavage and causes hypophosphatemia in vivo.

FGF-23 is involved in the pathogenesis of two similar hypophosphatemic diseases, autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and tumor-induced osteomalacia (TIO). We have shown that the overproduction of FGF-23 by tumors causes TIO. In contrast, ADHR derives from missense mutations in FGF-23 gene. However, it has been unclear how those mutations affect phosphate metabolism. Therefore, we produced mutant as well as wild-type FGF-23 proteins and examined their biological activity. Western blot analysis using site-specific antibodies showed that wild-type FGF-23 secreted into conditioned media was partially cleaved between Arg(179) and Ser(180). In addition, further processing of the cleaved N-terminal portion was observed. In constrast, mutant FGF-23 proteins found in ADHR were resistant to the cleavage. In order to clarify which molecule has the biological activity to induce hypophosphatemia, we separated full-length protein, the N-terminal and C-terminal fragments of wild-type FGF-23. When the activity of each fraction was examined in vivo, only the full-length FGF-23 decreased serum phosphate. Mutant FGF-23 protein that was resistant to the cleavage also retained the activity to induce hypophosphatemia. The extent of hypophosphatemia induced by the single administration of either wild-type or the mutant full-length FGF-23 protein was similar. In addition, implantation of CHO cells expressing the mutant FGF-23 protein caused hypophosphatemia and the decrease of bone mineral content. We conclude that ADHR is caused by hypophosphatemic action of mutant full-length FGF-23 proteins that are resistant to the cleavage between Arg(179) and Ser(180).

Integration of pre-normalized microarray data using quantile correction.

An enormous amount of microarray data has been collected and accumulated in public repositories. Although some of the depositions include raw and processed data, significant parts of them include processed data only. If we need to combine multiple datasets for specific purposes, the data should be adjusted prior to use to remove bias between the datasets. We focused on a GeneChip platform and a pre-processing method, RMA, and examined simple quantile correction as the post-processing method for integration. Integration of the data pre-processed by RMA was evaluated using artificial spike-in datasets and real microarray datasets of atopic dermatitis and lung cancer. Studies using the spike-in datasets show that the quantile correction for data integration reduces the data quality at some extent but it should be acceptable level. Studies using the real datasets show that the quantile correction significantly reduces the bias. These results show that the quantile correction is useful for integration of multiple datasets processed by RMA, and encourage effective use of public microarray data.

FGF-23 transgenic mice demonstrate hypophosphatemic rickets with reduced expression of sodium phosphate cotransporter type IIa.

Fibroblast growth factor (FGF)-23 was identified as a causative factor of tumor-induced osteomalacia and also as a responsible gene for autosomal dominant hypophosphatemic rickets. To clarify the pathophysiological roles of FGF-23 in these diseases, we generated its transgenic mice. The transgenic mice expressing human FGF-23 reproduced the common clinical features of these diseases such as hypophosphatemia probably due to increased renal phosphate wasting, inappropriately low serum 1,25-dihydroxyvitamin D level, and rachitic bone. The renal phosphate wasting in the transgenic mice was accompanied by the reduced expression of sodium phosphate cotransporter type IIa in renal proximal tubules. These results reinforce the notion that the excessive action of FGF-23 plays a causative role in the development of several hypophosphatemic rickets/osteomalacia.