Heterogeneity of colorectal cancers and extraction of discriminator gene signatures for personalized prediction of prognosis.

Dissected specimens of colorectal cancer (CRC) have been intensively studied using molecular sketches (gene signatures) to obtain a set of discriminator gene signatures for accurate prognosis prediction in individual patients. The discriminators obtained so far are not universally applicable, as the gene sets reflect the method and site of the study. In this study, we show that dissected stage II and III CRC samples are significantly heterogeneous in molecular sketches, and are not appropriate sources for discriminator extraction unless handled individually. To search for an accurate discriminator gene set for prediction of metastases, we need to start with less heterogeneous stage II CRC. We examined 198 (92 stage II and 106 stage III) CRC dissected samples for the predictability of discriminator gene signatures by analyzing stage II CRC alone, stage III alone, or in combination. The best predictive power of discriminator genes was obtained only when these genes were extracted and validated with stage II CRC samples. An accurate discriminator gene set for the prediction of CRC metastases can be obtained by focusing on stage II CRC samples.

Integration of cap analysis of gene expression and chromatin immunoprecipitation analysis on array reveals genome-wide androgen receptor signaling in prostate cancer cells.

The androgen receptor (AR) is a critical transcriptional factor that contributes to the development and the progression of prostate cancer (PCa) by regulating the transcription of various target genes. Genome-wide screening of androgen target genes provides useful information to understand a global view of AR-mediated gene network in PCa. In this study, we performed 5'-cap analysis of gene expression (CAGE) to determine androgen-regulated transcription start sites (TSSs) and chromatin immunoprecipitation (ChIP) on array (ChIP-chip) analysis to identify AR binding sites (ARBSs) and histone H3 acetylated (AcH3) sites in the human genome. CAGE determined 13 110 distinct, androgen-regulated TSSs (P<0.01), and ChIP-chip analysis identified 2872 androgen-dependent ARBSs (P<1e-5) and 25 945 AcH3 sites (P<1e-4). Both androgen-regulated coding genes and noncoding RNAs, including microRNAs (miRNAs) were determined as androgen target genes. Besides prototypic androgen-regulated TSSs in annotated gene promoter regions, there are many androgen-dependent TSSs that are widely distributed throughout the genome, including those in antisense (AS) direction of RefSeq genes. Several pairs of sense/antisense promoters were newly identified within single RefSeq gene regions. The integration of CAGE and ChIP-chip analyses successfully identified a cluster of androgen-inducible miRNAs, as exemplified by the miR-125b-2 cluster on chromosome 21. Notably, the number of androgen-upregulated genes was larger in LNCaP cells treated with R1881 for 24 h than for 6 h, and the percentage of androgen-upregulated genes accompanied with adjacent ARBSs was also much higher in cells treated with R1881 for 24 h than 6 h. On the basis of the Oncomine database, the majority of androgen-upregulated genes containing adjacent ARBSs and CAGE tag clusters in our study were previously confirmed as androgen target genes in PCa. The integrated high-throughput genome analyses of CAGE and ChIP-chip provide useful information for elucidating the AR-mediated transcriptional network that contributes to the development and progression of PCa.

[The malignant tumor of the pulmonary artery; report of a case].

Primary sarcoma of the pulmonary artery is rare. Diagnosis is difficult and often delayed; pulmonary embolism is part of the differential diagnosis. We treated a 46-year-old woman with progressive dyspnea. Computed tomography showed a lesion occupying the main pulmonary artery and peripheral branches on both sides. The pulmonary artery tumor, resected under cardiopulmonary bypass, was diagnosed pathologically as intimal sarcoma. Two weeks after the operation, the patient was feeling well and discharged from our hospital. Although the prognosis of pulmonary artery sarcoma is poor, early diagnosis and resection may prolong survival.

Effects of CO2 concentration and light intensity on photosynthesis of a rootless submerged plant, Ceratophyllum demersum L., used for aquatic food production in bioregenerative life support systems.

In addition to green microalgae, aquatic higher plants are likely to play an important role in aquatic food production modules in bioregenerative systems for producing feed for fish, converting CO2 to O2 and remedying water quality. In the present study, the effects of culture conditions on the net photosynthetic rate of a rootless submerged plant, Ceratophyllum demersum L., was investigated to determine the optimum culture conditions for maximal function of plants in food production modules including both aquatic plant culture and fish culture systems. The net photosynthetic rate in plants was determined by the increase in dissolved O2 concentrations in a closed vessel containing a plantlet and water. The water in the vessel was aerated sufficiently with a gas containing a known concentration of CO2 gas mixed with N2 gas before closing the vessel. The CO2 concentrations in the aerating gas ranged from 0.3 to 10 mmol mol-1. Photosynthetic photon flux density (PPFD) in the vessel ranged from 0 (dark) to 1.0 mmol m-2 s-1, which was controlled with a metal halide lamp. Temperature was kept at 28 degrees C. The net photosynthetic rate increased with increasing PPFD levels and was saturated at 0.2 and 0.5 mmol m-2 s-1 PPFD under CO2 levels of 1.0 and 3.0 mmol mol-1, respectively. The net photosynthetic rate increased with increasing CO2 levels from 0.3 to 3.0 mmol mol-1 showing the maximum value, 75 nmol O2 gDW-1 s-1, at 2-3 mmol mol-1 CO2 and gradually decreased with increasing CO2 levels from 3.0 to 10 mmol mol-1. The results demonstrate that C. demersum could be an efficient CO2 to O2 converter under a 2.0 mmol mol-1 CO2 level and relatively low PPFD levels in aquatic food production modules.

Isolation of Fusarium solani from a dog: identification by molecular analysis.

A strain of Fusarium solani was isolated from a dog showing many cutaneous and submucosal nodules and pyogranulomatous kidney lesions. Clinical isolates from this systemic infection were identified using microscopic examination and confirmed by molecular analysis.

Hematologic abnormalities and outcome of 16 cats with myelodysplastic syndromes.

We investigated the hematologic abnormalities and prognoses in 16 cats with myelodysplastic syndromes (MDS). Nonregenerative anemia, thrombocytopenia, and neutropenia were observed in 15, 13, and 4, respectively, of the 16 cats with MDS. Morphologic abnormalities characteristic of MDS included megaloblastoid rubricytes (9 cats), hyposegmentation of neutrophils (7 cats), nuclear abnormality of rubricytes (10 cats) and neutrophils (13 cats), and micromegakaryocytes (10 cats). Disease in these 16 cats was subclassified into refractory anemia (RA; 8 cats), RA with excess of blasts (RAEB; 5 cats), RAEB in transformation (RAEB in T; 1 cat), and chronic myelomonocytic leukemia (CMMoL; 2 cats), according to the human French-American-British (FAB) classification. In the cats in which the clinical outcome was known, 3 of 6 cats with high blast cell count MDS, including RAEB, RAEB in T, and CMMoL, developed acute myeloid leukemia, but only 1 of 8 cats with low blast cell count MDS (RA) developed acute myeloid leukemia. Based on the Dusseldorf scoring system for the prognosis of human MDS, the survival times of the cats showing high scores (> or =3 points) were significantly shorter than those of the cats with low scores (<3 points). The FAB classification and Dusseldorf scoring system were considered to be useful for predicting the prognosis of feline MDS. Furthermore, 15 of the 16 cats with MDS in this study were infected with feline leukemia virus, indicating its possible etiologic role in the pathogenesis of feline MDS.

4-Alkoxy-2-hydroxybenzaldehyde (AHB): a versatile aldehyde linker for solid-phase synthesis of C-terminal modified peptides and peptidomimetics.

[reaction: see text] A new and versatile 4-alkoxy-2-hydroxybenzaldehyde (AHB) linker for solid-phase syntheses is described. Acylation of the polymer-bound secondary amine obtained from reductive amination of the aldehyde in the AHB linker showed good reactivity. Following acylation of the phenolic hydroxyl group, the resulting carboxamide resin was stable to treatment with 95% TFA. The O-acyl functional group was removed with 20% piperidine and the desired compound was cleaved from the resin by TFA treatment.

Interaction of paratropomyosin with beta-connectin and its 400-kilodalton fragment from chicken skeletal muscle as influenced by the calcium ion concentration.

The binding of paratropomyosin to beta-connectin, which has been suggested to interact at the A-I junction of a sarcomere, was confirmed by measuring the changes in turbidity of a mixture with changing NaCl concentration, pH and free calcium ions, and by morphological observation and a coprecipitation assay of the aggregates formed in the mixture. Paratropomyosin also bound to the 400-kDa fragment which is the N-terminal portion of beta-connectin and contains the A-I junction region. Moreover, the interaction of paratropomyosin with the 400-kDa fragment was enhanced by a calcium ion concentration from 10(-7) M to 10(-5) M and markedly suppressed above 10(-4) M calcium ions. We conclude that paratropomyosin probably binds to the 400-kDa fragment of beta-connectin in the A-I junction region in living and pre-rigor skeletal muscle. In postmortem skeletal muscle paratropomyosin may be released from the 400-kDa portion of the connectin filament by increased calcium ion concentration and translocated on to thin filaments to induce meat tenderization.

Identification of a member of the serralysin family isolated from a psychrotrophic bacterium, Pseudomonas fluorescens 114.

An extracellular metalloprotease named No. 114 protease is one of the major secretions of a psychrotrophic bacterium, Pseudomonas fluorescens 114, the cold-adaptation mechanism of which has not been identified. In this study, we purified and cloned No. 114 protease, which is a single polypeptide having a molecular mass of 47 kDa. This protease contains a zinc-binding motif (HEXXHXUGUXH: X, arbitrary amino acid; U, bulky hydrophobic amino acid), glycine-rich repeats (GGXGXD) and no cysteine residue, which are the features specifically found in serralysin subfamily. No. 114 protease has its maximum activity at the temperature of 35-40 degrees C, which is about 20 degrees C lower than that of a serralysin from a mesophilic bacterium, Pseudomonas aeruginosa. All these results imply that No. 114 protease from this psychrophilic bacterium is a unique member of the serralysin group characterized by a low optimal temperature.

A low rate of nucleotide changes in Escherichia coli K-12 estimated from a comparison of the genome sequences between two different substrains.

Two genome sequences of Escherichia coli K-12 substrains, one partial W3110 and one complete MG1655, have been determined by Japanese and American genome projects, respectively. In order to estimate the rate of nucleotide changes, we directly compared 2 Mb of the nucleotide sequences from these closely-related E. coli substrains. Given that the two substrains separated about 40 years ago, the rate of nucleotide changes was estimated to be less than 10(-7) per site per year. This rate was supported by a further comparison between partial genome sequences of E. coli and Shigella flexneri.

Topology selection in unrooted molecular phylogenetic tree by minimum model-based complexity method.

In reconstruction of phylogenetic trees from molecular data, it has been pointed out that multifurcate phylogenetic trees are difficult to be correctly reconstructed by the conventional methods like maximum likelihood method(ML). In order to resolve this problem, we have been engaged in developing a new phylogenetic tree reconstruction method, based on the minimum complexity principle widely used in the inductive inference. Our method, which we call "minimum model-based complexity (MBC) method", has been proved so far to be efficient in estimating multifurcate branching when the tree is described in the form of rooted one. In this study, we make further investigations about the efficiency of MBC method in estimating the multifurcation in unrooted phylogenetic trees. To do so, we conduct computer simulation in which the estimations by MBC method are compared with those by ML, AIC and statistical test approach. The results show that MBC method also provides good estimations even in the case of multifurcate unrooted trees and suggest that it could be generally used for reconstruction of phylogenetic tree having arbitrary multifurcations.

Exploring the structure-activity relationships of [1-(4-tert-butyl-3'-hydroxy)benzhydryl-4-benzylpiperazine] (SL-3111), a high-affinity and selective delta-opioid receptor nonpeptide agonist ligand.

SL-3111 [1-(4-tert-butyl-3'-hydroxy)benzhydryl-4-benzylpiperazine] is a de novo designed, high-affinity and selective nonpeptide peptidomimetic agonist of the delta-opioid receptor. In a previous report we had described the unique biological characteristics of this ligand and also a need for further structural evaluation.(6) To pursue this, we have introduced a completely different heterocyclic template (2 and 3), which, based on molecular modeling studies, may present the required structural features to properly orient the pharmacophore groups. We also have made more subtle changes to the original piperazine scaffold (5 and 11). The biological activities of these compounds revealed an important participation of the scaffold in the ligand-receptor interaction. To further explore functional diversity on the scaffold, we have maintained the original piperazine ring and introduced four different functionalities at position 2 of the heterocyclic ring (15a-d; a = CH(2)-O-CH(2)-Ph; b = Me; c = CH(2)Ph; d = CH(2)OH). The biological activities observed for these compounds showed a very interesting trend in terms of the steric effects of the groups introduced at this position. A decrease of almost 2000-fold in affinity and potency at the delta-receptor was observed for 15c compared with 15b. This difference may be explained if we postulate that the bioactive conformation of these peptidomimetics is close to the minimal energy conformations calculated in our study. On the basis of these findings we have realized the importance of this position to further explore and simplify the structure of future generations of peptidomimetic ligands.

Formal design and implementation of an improved DDBJ DNA database with a new schema and object-oriented library.

MOTIVATION: The DNA Data Bank of Japan (DDBJ) has developed a new DNA database system with a new schema design to accommodate rapid change and growth of requirements on the system. RESULTS: The new schema and systems were created using an object-oriented design approach. The design was accomplished in accordance with ANSI/SPARC three-level schema architecture. First, the conceptual schema was designed using a functional model named AIS (associative information structure) and was visualized in extended diagram format. The model is a natural extension of an ER (entity relationship) model and describes real-world objects in binary associations between entities with the concept of order. Second, the schema was mapped on a relational database as a physical schema. All details are concentrated in this schema and the layer lying above enjoys physical independence. Finally, as another layer, external modeling was introduced for the database applications interface. It provides set-at-a-time basis operations and was implemented as a C++ object-oriented library. On this common framework of a new schema, a new annotator's workbench named Yamato II and a World Wide Web (WWW) submission system named Sakura have been successfully developed to improve drastically daily transactions in the DDBJ. AVAILABILITY: Sakura is available at the following address: http://sakura.ddbj.nig.ac.jp. CONTACT: hsugawar@genes.nig.ac.jp

Binding of paratropomyosin to beta-connectin from chicken skeletal muscle.

We found that paratropomyosin bound to beta-connectin, in examining binding of paratropomyosin at the junction of A- and I-bands of sarcomeres. The turbidity of a mixture of beta-connectin and paratropomyosin was greater with more paratropomyosin added, but high concentrations of Ca2+ suppress this increase. These results suggest that paratropomyosin is released from connectin filaments at the A-I junction region by increased concentrations of calcium ions in postmortem skeletal muscles.

The application of model-based complexity inference method to molecular evolution analysis.

In this study, a new method based on the concept of complexity in inductive inference is proposed for reconstructing molecular phylogenetic tree. This method describes the complexity of molecular phylogenetic tree by three terms, which are related to tree topology, the branch lengths and fitness between the model and data measured by likelihood function. The computer simulation is used to investigate the efficiency of this method. The results suggest that this method is superior to the traditional methods because it avoids excess-complexity in the tree model estimation available from DNA sequence.

Anticoagulant activity of the novel thrombin inhibitor 1-butyl-3-(6,7-dimethoxy-2-naphthylsulfonyl) amino-3-(3-guanidinopropyl)-2-pyrrolidinone hydrochloride.

1-Butyl-3-(6,7-dimethoxy-2-naphthylsulfonyl)amino-3-(3-guanidin opropyl)-2-pyrrolidinone hydrochloride (CAS 173440-64-7, SPI-501), which has highly selective thrombin-inhibitory activity, caused a concentration-dependent increase in the time taken for coagulation induced by thrombin in rabbit plasma. The IC50 of SPI-501 was 1.7 mumol/l. Argipidine also prolonged coagulation time and its activity was one order of magnitude greater than that of SPI-501. SPI-501 and argipidine caused dose-dependent increases in the activated partial prothrombin time (APTT) and prothrombin time (PT) of rat plasma. When APTT and PT were measured, IC50 values of SPI-501 were 38.0 and 18.5 mumol/l and those of argipidine, 1.4 and 1.9 mumol/l, respectively. Intravenous administration of SPI-501 (10 and 30 mumol/kg) and argipidine (1 and 3 mumol/kg) prolonged both APTT and PT in rats. While SPI-501 was less potent than argipidine, the durations of the effects of both were the same.

Antithrombotic effects of NE-6505, a novel anion-binding exosite inhibitor.

NF-6505, a bi-O-Tyr-sulfated decapeptide, which specifically interacts with the anion-binding exosite of the thrombin molecule, was chemically synthesized and assessed for its antithrombotic effects in vitro and in vivo. The IC50 value of this peptide on fibrin-clot formation in vitro was about 0.05 microgram/ml, which indicated a potency similar to that of a recombinant hirudin. NF-6505 caused a 2-fold prolongation of activated partial thromboplastin time when intravenously administered at 1 mg/kg in rats. In a rat venous thrombosis model, a bolus intravenous administration of this peptide dose-dependently inhibited the thrombus formation with an ED50 value of 0.03 mg/kg, a value smaller than that of recombinant hirudin (ED50 = 0.1 mg/kg) or of argatroban (ED50 = 0.2 mg/kg). These results suggest that NF-6505 is a highly potent and safe agent for the clinical treatment of venous thrombosis diseases.

Antithrombotic effects of the novel inhibitor of thrombin-induced offtelet aggregation and thrombus formation, 3-[2-[1,1':2',1"]-terphenyl-4'-yl)ethyl]phenoxyacetic acid.

The new compound 3-[2-([1,1':2,1"]-terphenyl-4'yl)ethyl]phenoxyacetic acid (F1070) was synthesized and its effects on platelet aggregation induced by thrombin, thrombin receptor agonist peptide (TRAP), ADP and collagen were evaluated in humans, guinea pigs and rats, and were compared with the effects of he thrombin antagonists argipidine and (D)Phe-Pro-Arg-CH2Cl (FPR). F1070 inhibited the platelet aggregation induced by these agonists and was highly selective in its inhibition of thrombin. F1070 inhibited fibrin formation induced by thrombin, but far less effectively than argipidine. In a guinea pig model of extracorporeal circulation thrombosis, F1070 (10 mg/kg p.o.) significantly inhibited the development of a thrombus. F1070 is thus a key compound that should facilitate the development of new orally active antithrombotic drugs that are specific for thrombin.

Inference of molecular phylogenetic tree based on minimum model-based complexity method.

In this study, starting with a newly introduced concept of data complexity ("empirical data complexity"), we specify the concept of complexity more concretely in relation to mathematical modeling and introduce "model-based complexity (MBC)". Inductive inference based on the minimum model-based complexity method is then applied to the reconstruction of molecular evolutionary tree from DNA sequences. We find that minimum MBC method has good asymptotic property when DNA sequence lengths approach to infinite and compensates the bias of maximum likelihood method due to the difference of tree topology complexity. The efficiency of minimum MBC method for reconstruction of molecular tree is studied by computer simulation, and results suggest that this method is superior to the traditional maximum likelihood method or its modification by Akaike's AIC.

Structure-activity studies on C-terminal hirudin peptides containing sulfated tyrosine residues.

To clarify the role of the negative charge of the C-terminal region of hirudin, we chemically synthesized the C-terminal peptide of hirudin variant-1 (HV-1), HV-1-(54-65), and its analogs, [E61Y,E62Y]HV-1-(54-65) and [E62Y]HV-1-(54-65), and then sulfated the Tyr residue(s) in these peptides by both enzymic and chemical methods. Enzymic O-sulfation of Tyr residues in the peptides by use of sulfotransferase isolated from Eubacterium A-44 allowed us to produce four kinds of the sulfated peptide, whose C-terminal sequences were -PEY(SO3H)YLQ, -PYY(SO3H)YLQ, -PYYY(SO3H)LQ and -PYY(SO3H)Y(SO3H)LQ. On the other hand, all Tyr residues in the peptides were successfully sulfated by chemical reaction with N,N'-dicyclohexylcarbodiimide in the presence of sulfuric acid. Based on the analysis of structure-activity relationships of these sulfated peptides for thrombin inhibition, the Tyr62 and Tyr63 bisulfated peptide GDFEEIPEY(SO3H)Y(SO3H)LQ was found to be the most potent inhibitor of thrombin among the products tested. No increase in potency was observed by further substitution of Glu61 with Tyr(SO3H). The inhibitory activity by substitution with Tyr(SO3H) at position 63 was greater than that obtained by the substitution at position 62.