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M.-A. von Mach
J. G. Hengstler
Leibniz Research Centre for Working Environment and Human Factors
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L. S. Weilemann, Mainz, Germany
I. Ishibashi, Tokyo, Japan
P. Micke, Uppsala, Sweden
J. Timmer, Freiburg, Germany
A. Winterpacht, Erlangen, Germany
B. C. Behera, Pune, India

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Recognition of DNA Splice Junction via Machine Learning Approaches
(2005-10-14) Isarankura-Na-Ayudhya, Chartchalerm; Naenna, Thanakorn; Nantasenamat, Chanin; Prachayasittikul, Virapong
Successful recognition of splice junction sites of human DNA sequences was achieved via three machine learning approaches. Both unsupervised (Kohonen's Self-Organizing Map, KSOM) and supervised (Back-propagation Neural Network, BNN; and Support Vector Machine, SVM) machine learning techniques were used for the classification of sequences from the testing set into one of three categories: transition from exon to intron, transition from intron to exon, and no transition. The dataset used in this study is comprised of 1,424 DNA sequences obtained from the National Center for Bioinformatics Information (NCBI). Performance of the machine learning approaches were assessed by the construction of learning models from 1,000 sequences of the training set and evaluated on the 424 sequences of the testing set that is unknown to the learning model. Each sequence is a window of 32 nucleotides long with regions comprising -15 to +15 nucleotides from the dinucleotide splice site. Since the nucleotides (A, C, G, and T) are represented by four digit binary code (e.g. 0001, 0010, 0100, and 1000) the number of descriptors increased from 32 to 128. The performance of machine learning techniques in order of increasing accuracy are as follows SVM > BNN > KSOM, suggesting that SVM is a robust method in the identification of unknown splice site. Although KSOM gave lower prediction accuracy than the two supervised methods, it is fascinating that it was able to make such prediction based only on knowledge of the input whereas the supervised method requires that the output be known during training. It is expected that the Support Vector Machine method can provide a powerful computational tool for predicting the splice junction sites of uncharacterized DNA.
Expression of the protein phosphatases PP1a and PP1g1 in rat ascites hepatoma cell lines and in rat hepatocyte cell line
(2005-10-10) Kikuchi, Kunimi; Saadat, Mostafa
Expressions of protein phosphatases 1a (PP1a), and 1g1 (PP1g1) was determined in cultured rat ascites hepatoma cell lines (AH-13, AH-109A) and in the normal rat hepatocyte cell line RLN-B2 by Northern blot analysis. High levels of PP1a mRNA expression were observed only in the hepatoma cells. In RLN-B2 cells only PP1g1 mRNA was increased compared to normal rat liver. In all cell types, mRNA levels of PP1g1 decreased as a function of harvest time. The present data show that PP1a mRNA is expressed at high levels in hepatoma cells in vivo, but rapidly decreased under critical nutritional conditions.
Construction of chimeric antibody binding green fluorescent protein for clinical application
(2005-10-07) Isarankura-Na-Ayudhya, Chartchalerm; Prachayasittikul, Virapong; Suwanwong, Yaneenart; Tantimavanich, Srisurang
A chimeric antibody-binding green fluorescent protein (ZZGFPuv) was successfully constructed and applied as a powerful tool for immunological diagnosis. A gene encoding two repetitive sequences of Z-domain, derivative of IgG-binding B domain of staphylococcal protein A, was fused in-frame to the N-terminus of gfpuv gene. The chimeric gene was subsequently transformed and expressed in various strains of E. coli. Expression of chimeric protein in E. coli strain HB101 resulted in a protein translocation from cytoplasm to periplasmic space and cultivation medium. The chimeric ZZGFPuv could be purified using either IgG Sepharose column or immobilized metal (Cu2+) affinity chromatography. The purified protein migrated in non-denaturing SDS-PAGE as two major bands. A fluorescent band was located at 36 kDa while another band at 48 kDa exhibited non-fluorescence. The fluorescent band was isolated and assessed for IgG-binding via fluorescent emission. The lowest amount of IgG that could be detected by dot immunobinding assay was approximately 630 ng. Indirect immunofluorescent assay for a serological detection of leptospirosis was performed by using the chimeric ZZGFPuv as IgG detector. A strong fluorescent intensity as comparable to that of the fluorescein isothiocyanate (FITC) conjugated system was significantly detected. All these findings support a high feasibility to apply the chimeric Ab-binding GFP for clinical applications in the future.
Biological and structural characterizations of mutations in X-linked spondyloepiphyseal dysplasia tarda
(2006-09-16) Jang, Se Bok; Jeong, Mi Suk; Lee, Kyoung Mi
Spondyloepiphyseal dysplasia tarda (SEDT), an X-linked genetic disease manifesting itself in a disproportionate skeletal structure, is caused by mutations in the SEDL gene. Four missense mutations (S73L, V130D, F83S, and D47Y) have been identified by molecular diagnosis as disease-causing SEDT. Nevertheless, how SEDL mutations disrupt the skeletal structure and cause disease remains unknown. We report here the cloning, expression, and characterization of three different missense mutations (S73L, V130D, and D47Y) in mouse SEDL. The overexpression of the D47Y mutation was mainly observed in the supernatant but those of the S73L and V130D mutations are shown in the insoluble pellets. The substitution of the S73L mutation induces the exposure to hydrophobic amino acids and causes aggregation. That of V130D might break hydrophobic interaction and decrease the secondary structure. The CD spectra of three mutants (S73L, V130D, and D47Y) showed that the a-helices decreased more than that of wild-type SEDL. The F83S (stop) mutant might suggest a large conformational change as the mutant V130D. In order to visualize conformational changes in mutated structures, we used molecular modeling techniques minimizing the total energy. These results could provide the biological characterization and conformational information of the SEDL mutants and suggest the clinical severity of the disorder among human SEDL patients.
IL-10 Inhibits Transforming Growth Factor-ß-Induction of Type I Collagen mRNA Expression via Both JNK and p38 Pathways in Human Lung Fibroblasts
(2005-08-11) Arai, Toru; Hirata, Haruhiko; Hoshino, Shigenori; Inoue, Koji; Kawase, Ichiro; Kida, Hiroshi; Kijima, Takashi; Kumagai, Toru; Osaki, Tadashi; Tachibana, Isao; Takimoto, Takayuki; Yanagita, Masahiko; Yano, Yukihiro; Yoshida, Mitsuhiro
Transforming growth factor-ß (TGF-ß) is a key factor for understanding the pathogenesis of fibrotic disorders such as idiopathic pulmonary fibrosis (IPF). We have demonstrated that interleukin-10 (IL-10) suppresses TGF-ß-induced expression of type I collagen (COL1) mRNA in a human lung fibroblast cell line (WI-38). However, the inhibitory mechanism has not yet been clearly elucidated. Thus, in the current study, we investigate the effects of IL-10 blockade of TGF-ß signaling which regulates COL1 mRNA expression. In WI-38 cells, IL-10 inhibits TGF-ß-mediated phosphorylation of both, c-Jun HN2-terminal kinase (JNK) and p38, but does not suppress TGF-ß- mediated phosphorylation of Smad2 or affect TGF-ß-upregulation of Smad7 mRNA expression. In addition, SP600125 and SB203580, specific inhibitors of JNK and p38, respectively, attenuate TGF-ß-induced COL1 mRNA expression in WI-38 cells. These results suggest that IL-10 inhibits TGF-ß-induced COL1 mRNA expression via both JNK and p38 pathways but not Smad pathways in WI-38 cells. This inhibitory mechanism may provide a novel insight into therapeutic strategies for fibrotic disorders such as IPF.
Stage-specific expressions of four different ribonuclease H genes in Leishmania
(2005-07-13) Chaudhuri, Gautam; Farrow, Anitra L.; Misra, Smita
The human pathogen of the genus Leishmania cause worldwide morbidity and infection of visceral organs by some species may be lethal. Lack of rational chemotherapy against these pathogens dictates the study of differential biochemistry and molecular biology of the parasite as compared to its human host. The ubiquitous enzyme ribonuclease H (RNase H, EC 3.1.26.4) cleaves the RNA from a RNA:DNA duplex and is critical for the replication of DNA in the nucleus and the mitochondria. We have characterized four RNase H genes from Leishmania: one is of type I (LRNase HI) and three others are of type II (LRNase HIIA, -HIIB and -HIIC). In contrast human cells have only one type I and one type II RNase H. All the four RNase H genes in Leishmania are single copy and located in discrete chromosomes. When expressed inside RNase H-deficient E. coli, all of the four Leishmania RNase H were capable to complement the genetic defect of the E. coli, indicating their identity as RNase H. The enzymes are differentially expressed in the promastigotes and the amastigotes, the forms that thrives in entirely different physico-chemical conditions in nature. Nucleotide sequences of the 5'-UTRs of three of these mRNAs have upstream open reading frames. Understanding the regulation of these four distinct ribonuclease H genes in Leishmania will help us better understand leishmanial parasitism and may help us to design rational chemotherapy against the pathogen.
Morphological and genetic characteristics of Nicotiana langsdorffii, N. glauca and its hybrid
(2006-07-04) Han, Woong; Heo, Kweon; Jin, Ying-Shan; Lim, Hak-Tae; Wang, Myeong-Hyeon
Plant tumors, including genetic tumors, are disorganized and proliferate in an uncontrolled fashion. In this report we describe the morphological, physiological and genetic properties of Nicotiana. langsdorffii and Nicotiana. glauca and their hybrids (Nicotiana. langsdorffii x Nicotiana. Glauca). Nicotiana. langsdorffii leaves are oblate and pubescent with winged petioles, while Nicotiana. glauca leaf-blades are rubbery and oval-to-heart-shaped. The hybrid plants are intermediate in leaf shape, with anisocytic stomata and well-developed trichomes. In addition, they produce tumors in the absence of bacteria and exogenous hormones. Tumor growth in the hybrid plants was not affected by indole-3-acetic acid or kinetin. Genetic polymorphism was analyzed by the randomly amplified polymorphic DNA technique in the parents (Nicotiana. langsdorffii and Nicotiana. glauca) and in the genetic tumors produced by the hybrids. A total of 128 randomly amplified polymorphic DNA fragments were scored from fifteen random primers, and pronounced differences were found between the genetic tumors and their parents. These observations show that randomly amplified polymorphic DNA markers are might informative about genetic similarities and dissimilarities.
The effects of aB-crystallin on mitochondrial death pathway during hydrogen peroxide induced apoptosis
(2005-06-20) Deng, Gonghua; Li, Junli; Liu, Meidong; Liu, Shuang; Tu, Zizhi; Xiao, Xianzhong
aB-crystallin, a major small heat shock protein, has recently been shown to exert inhibitory effects on apoptosis, while the responsible mechanisms remain largely unknown. In the present study, we discovered that aB-crystallin protected mouse myoblast C2C12 cells against oxidative stress-induced apoptosis. During hydrogen peroxide-induced apoptosis, aBcrystallin showed that it decreased the redistribution level of phosphatidylserine (PS), reduced the release of cytochrome C and Smac/Diablo from mitochondria into cytoplasm, and decreased the cleavage of Bid. Interestingly, immunoprecipitation experiments with anti-aBcrystallin and anti-myc-tag antibodies demonstrated respectively an interaction between aBcrystallin and p53 during hydrogen peroxide induced apoptosis. Both the NH2-terminal and COOH-terminal regions of aB-crystallin could interact with p53, suggesting two domains of aB-crystallin are necessary for the interaction. Electrophoresis mobility shift assay (EMSA) and luciferase assay further demonstrated that aB-crystallin inhibited the upregulation of the DNA-binding, as well as the transactivation activity of p53 induced by hydrogen peroxide. Our results show that aB-crystallin has a protective role in oxidative stress induced apoptosis by interference with the mitochondrial death pathway.
Expression of prostacyclin-stimulating factor (PSF) in mononuclear cells of human peripheral blood and THP-1 derived macrophage-like cells, and effects of high glucose concentration
(2005-12-20) Etoh, Takashi; Hashimoto, Toshihiko; Imamura, Minako; Inoguchi, Toyoshi; Nawata, Hajime; Sekiguchi, Naotaka
Prostacyclin (PGI2) synthesis by vascular endothelial cells (ECs) decreases in diabetic subjects, possibly leading to development of diabetic angiopathies including that in atherosclerosis. We identified a bioactive peptide, prostacyclin-stimulating factor (PSF), which stimulates PGI2 synthesis in cultured aortic ECs. Our previous studies demonstrated that PSF was predominantly expressed by arterial smooth muscle cells (SMCs) and ECs. We immunohistochemically showed that PSF existed in SMCs of human coronary arteries, and PSF staining was markedly reduced in coronary arterial SMCs of patients with type 2 diabetes and/or myocardial infarction. In the present study, we investigated the existence of PSF in human serum, and effects of glucose on serum PSF levels in patients with type 2 diabetes. Immunoblot analysis revealed the presence of PSF in serum, and showed that serum PSF protein concentration was significantly decreased in type 2 diabetic patients. Moreover, there was a significant negative correlation between serum PSF and HbA1c levels in these patients. Using immunohistochemistry, we also showed that PSF was present in serum and in macrophages (Mfs). PSF mRNA was found in Mfs using reverse transcription-polymerase chain reaction (RT-PCR). In addition, effects of high glucose conditions on PSF production in Mfs were examined by Western blotting, and we showed that PSF significantly decreased when Mfs were cultured in high glucose conditions. These results strongly suggested that decreased PSF production might result in decreased production of PGI_2 in atherosclerotic lesions, thus leading to development of diabetic macroangiopathy and atherosclerosis.
Increasing trend of multiple resistance and genomic mobility of Neisseria gonorrhoeae to penicillin and quinolone
(2005-12-05) Buatiang, Anukul; Jittawoutipoka, Thichakorn; Lawung, Ratana; Prachayasittikul, Virapong; Rittiroongrad, Surawach
A significant decline of gonorrhea incidence has been observed during the years 1990-99. However, a slight increase in the number of cases has been reported in 2000. In addition, an increase in resistant strains has been found in Thailand. In this study, 207 isolates of N. gonorrhoeae from patients attending Bangrak hospital (National Centre of Sexually Transmitted Infections), 67 isolates obtained during January-March 2000, 74 isolates obtained during January-March 2002, and 66 isolates obtained during October-December 2002, were tested. All isolates were susceptible to ceftriaxone while 71.5% and 74.4% were resistant to penicillin and quinolone, respectively. The high level of ciprofloxacin resistance (MIC =4 mg/L) also increased from 13.4% during January-March 2000 to 25.8% during October-December 2002. Multiple resistance determinants commonly coexisted in a single isolate so that the level of resistance was increased. The incidence of double resistance determinants, penicillin and quinolone resistance, were significantly increased from 34.3% among isolates during January to March 2000 up to 77.3% among isolates during October to December 2002 (P < 0.001). In addition, an isolate obtained in 2002 resisted to spectinomycin with a high MIC (>1.024 g/L). Several plasmid patterns have been identified and various patterns of the plasmid can be artificially transferred and maintained their expression in Escherichia coli transformants. Such evidences infer the high mobility of resistant genome among microorganisms in the region. Moreover, the significant increase in penicillin and quinolone resistance herein, indicates the selective pressure and the diversity of genomic distribution among N. gonorrhoeae in Thailand. Primers JDA (5-TAC TCA ATC GGT AAT TGG CTT C-3) and JDB (5-CCA TAT CAC CGT CGG TAC TG-3) have been designed from sequences of the Asia, the Africa, and the Toronto ß-lactamase plasmids. By using the JDA and the JDB as PCR primers, our data reveal the highest prevalence and a significantly increasing trend of the epidemic Africa type of genomic ß-lactamase.
Strenuous physical exercise induces monocyte chemoattractant protein-1 release in patients with coronary artery disease
(2005-02-03) Becker, Dietmar; Lindemann, Stephan; Seul, Marco
Infiltration of the arterial vessel wall with monocytes is one of the initial inflammatory events. Monocyte chemoattractant protein-1 (MCP-1) is the key chemokine for the recruitment of monocytes to the atherosclerotic lesion. So far, it is unknown, if strenuous exercise enhances or reduces the release of MCP-1, the key initiator of pro-atherosclerotic inflammatory events in patients at risk for atherosclerotic diseases. 15 Patients with at least three coronary risk factors (CRF) like smoking, hypertension, diabetes, hypercholesterolemia and overweight and 17 corresponding healthy controls were tested with bicycle ergometry. Additionally, 8 patients with coronary artery disease (CAD) were investigated. Before and 10 minutes after maximal exercise, venous blood was taken and MCP-1 serum levels were analyzed. Furthermore, we measured monocyte CD11b expression by flow cytometry. Independently of CRF, the MCP-1 serum level was significantly increased after exercise. In control subjects the MCP-1 serum level rose from 71 pg/ml to 94 pg/ml (p<=0.05). In patients with CRF the MCP-1 serum level went up from 154 pg/ml to 224 pg/ml (p<=0.05). Patients with coronary artery disease had elevated MCP-1 serum levels before and after exercise, too, even if they did not have CRF (143 vs. 174 pg/ml; p<=0.05). The monocyte activation parameter CD11b showed a significant raise after physical exercise (relative fluorescence intensity 31 vs. 44; p<=0.05). These data indicate, that MCP-1 serum levels are elevated after physical exercise especially in patients at risk for coronary artery disease. These effects may in part result from an increased monocyte activation following strenuous physical exercise.

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