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1.  Life-Threatening Gastrointestinal Mucosal Necrosis during Methotrexate Treatment for Rheumatoid Arthritis 
Case Reports in Gastroenterology  2013;7(3):470-475.
Methotrexate (MTX), a folic acid antagonist, is widely used in the treatment of neoplasms, psoriasis and rheumatoid arthritis. Despite its efficacy, MTX sometimes finds limited application because of its adverse effects, including renal or liver impairment, bone marrow toxicity and gastrointestinal mucosal injury. Intestinal mucositis, bleeding and peptic ulcers are well-known gastrointestinal adverse effects of MTX, although cases of fatal mucosal necrosis induced by MTX are extremely rare. Here, we report the case of an 82-year-old Japanese woman who developed severe gastrointestinal mucosal necrosis after 8 years of treatment with low-dose MTX (8 mg/week). In the drug lymphocyte stimulation test, MTX showed a strong positive reaction, with a stimulation index of 443% against normal controls. Physicians must be aware of potential drug-induced adverse effects in patients with chronic diseases who are on long-term medication.
doi:10.1159/000356817
PMCID: PMC3843920  PMID: 24348319
Methotrexate; Adverse effect; Drug lymphocyte stimulation test; Type IV allergic reactions; Gastrointestinal tract; Mucosal injury; Necrosis; Rheumatoid arthritis
2.  Cholecystomucoclasis: revaluation of safety and validity in aged populations 
BMC Gastroenterology  2012;12:113.
Background
We evaluated the safety and validity of cholecystomucoclasis (CM) and compared its intraoperative characteristics with those of standard cholecystectomy (SC).
Methods
We enrolled 174 patients who underwent cholecystectomy and retrospectively evaluated the outcomes of patients in the SC and CM groups.
Results
Significant differences in age (71.1 vs. 61.9 years), American Society of Anesthesiologists physical status (ASA-PS), and serum C-reactive protein levels (CRP) (18.1 vs. 4.7 mg/dL) were observed between the CM and SC groups. Conversely, no significant differences were observed in the operation time (129 vs. 108 min), amount of blood loss (147 vs. 80 mL), intraoperative complications (0% vs. 5.7%), or duration of hospital stay (13.2 vs. 8.9 days) between the 2 groups. A high conversion rate (35.3%), postoperative complications (33%), and frequent drain insertions (94%) were observed in the CM group.
Conclusions
CM is a safe and valid surgical procedure and surgeons should not hesitate to transition to CM for patients who are of advanced age, in poor general condition (high ASA classification), or have high levels of serum CRP.
doi:10.1186/1471-230X-12-113
PMCID: PMC3462142  PMID: 22909056
Cholecystitis; Cholecystomucoclasis; Deroofing; Subtotal cholecystectomy
3.  MAFFT version 5: improvement in accuracy of multiple sequence alignment 
Nucleic Acids Research  2005;33(2):511-518.
The accuracy of multiple sequence alignment program MAFFT has been improved. The new version (5.3) of MAFFT offers new iterative refinement options, H-INS-i, F-INS-i and G-INS-i, in which pairwise alignment information are incorporated into objective function. These new options of MAFFT showed higher accuracy than currently available methods including TCoffee version 2 and CLUSTAL W in benchmark tests consisting of alignments of >50 sequences. Like the previously available options, the new options of MAFFT can handle hundreds of sequences on a standard desktop computer. We also examined the effect of the number of homologues included in an alignment. For a multiple alignment consisting of ∼8 sequences with low similarity, the accuracy was improved (2–10 percentage points) when the sequences were aligned together with dozens of their close homologues (E-value < 10−5–10−20) collected from a database. Such improvement was generally observed for most methods, but remarkably large for the new options of MAFFT proposed here. Thus, we made a Ruby script, mafftE.rb, which aligns the input sequences together with their close homologues collected from SwissProt using NCBI-BLAST.
doi:10.1093/nar/gki198
PMCID: PMC548345  PMID: 15661851
4.  Basal jawed vertebrate phylogeny inferred from multiple nuclear DNA-coded genes 
BMC Biology  2004;2:3.
Background
Phylogenetic analyses of jawed vertebrates based on mitochondrial sequences often result in confusing inferences which are obviously inconsistent with generally accepted trees. In particular, in a hypothesis by Rasmussen and Arnason based on mitochondrial trees, cartilaginous fishes have a terminal position in a paraphyletic cluster of bony fishes. No previous analysis based on nuclear DNA-coded genes could significantly reject the mitochondrial trees of jawed vertebrates.
Results
We have cloned and sequenced seven nuclear DNA-coded genes from 13 vertebrate species. These sequences, together with sequences available from databases including 13 jawed vertebrates from eight major groups (cartilaginous fishes, bichir, chondrosteans, gar, bowfin, teleost fishes, lungfishes and tetrapods) and an outgroup (a cyclostome and a lancelet), have been subjected to phylogenetic analyses based on the maximum likelihood method.
Conclusion
Cartilaginous fishes have been inferred to be basal to other jawed vertebrates, which is consistent with the generally accepted view. The minimum log-likelihood difference between the maximum likelihood tree and trees not supporting the basal position of cartilaginous fishes is 18.3 ± 13.1. The hypothesis by Rasmussen and Arnason has been significantly rejected with the minimum log-likelihood difference of 123 ± 23.3. Our tree has also shown that living holosteans, comprising bowfin and gar, form a monophyletic group which is the sister group to teleost fishes. This is consistent with a formerly prevalent view of vertebrate classification, although inconsistent with both of the current morphology-based and mitochondrial sequence-based trees. Furthermore, the bichir has been shown to be the basal ray-finned fish. Tetrapods and lungfish have formed a monophyletic cluster in the tree inferred from the concatenated alignment, being consistent with the currently prevalent view. It also remains possible that tetrapods are more closely related to ray-finned fishes than to lungfishes.
PMCID: PMC387836  PMID: 15070407
5.  MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform 
Nucleic Acids Research  2002;30(14):3059-3066.
A multiple sequence alignment program, MAFFT, has been developed. The CPU time is drastically reduced as compared with existing methods. MAFFT includes two novel techniques. (i) Homo logous regions are rapidly identified by the fast Fourier transform (FFT), in which an amino acid sequence is converted to a sequence composed of volume and polarity values of each amino acid residue. (ii) We propose a simplified scoring system that performs well for reducing CPU time and increasing the accuracy of alignments even for sequences having large insertions or extensions as well as distantly related sequences of similar length. Two different heuristics, the progressive method (FFT-NS-2) and the iterative refinement method (FFT-NS-i), are implemented in MAFFT. The performances of FFT-NS-2 and FFT-NS-i were compared with other methods by computer simulations and benchmark tests; the CPU time of FFT-NS-2 is drastically reduced as compared with CLUSTALW with comparable accuracy. FFT-NS-i is over 100 times faster than T-COFFEE, when the number of input sequences exceeds 60, without sacrificing the accuracy.
PMCID: PMC135756  PMID: 12136088
6.  Commitment and Differentiation of Osteoclast Precursor Cells by the Sequential Expression of C-Fms and Receptor Activator of Nuclear Factor κb (Rank) Receptors 
The Journal of Experimental Medicine  1999;190(12):1741-1754.
Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor κB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms+RANK−) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms+RANK+) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK–RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.
PMCID: PMC2195707  PMID: 10601350
osteoclastogenesis; commitment; macrophage; RANK ligand; M-CSF
7.  The Complete Nucleotide Sequence of the Human Immunoglobulin Heavy Chain Variable Region Locus  
The Journal of Experimental Medicine  1998;188(11):2151-2162.
The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified. The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of VH region was calculated to be ∼6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes. Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family. However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region.
PMCID: PMC2212390  PMID: 9841928
immunoglobulin gene; physical map; antibody repertoire; phylogenetic tree; DNA duplication
8.  When Overexpressed, a Novel Centrosomal Protein, RanBPM, Causes Ectopic Microtubule Nucleation Similar to γ-Tubulin  
The Journal of Cell Biology  1998;143(4):1041-1052.
A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with γ-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to γ-tubulin was faded by the addition of GTPγS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.
PMCID: PMC2132962  PMID: 9817760
centrosome; γ-tubulin; Ran; RanBPM; YGL227w

Results 1-8 (8)