Citrullinated inter-alpha-trypsin inhibitor heavy chain 4 in arthritic joints and its potential effect in the neutrophil migration.

The citrullinated inter-alpha-trypsin inhibitor heavy chain 4 (cit-ITIH4) was identified as its blood level was associated with the arthritis score in peptide glucose-6-phosphate-isomerase-induced arthritis (pGIA) mice and the disease activity in patients with rheumatoid arthritis (RA). This study aimed to clarify its citrullination pathway and function as related to neutrophils. In pGIA-afflicted joints, ITIH4 and cit-ITIH4 levels were examined by immunohistochemistry (IHC), immunoprecipitation (IP) and Western blotting (WB), while peptidylarginine deiminase (PAD) expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), IHC and immunofluorescent methods. The pGIA mice received anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies to deplete neutrophils and the expression of cit-ITIH4 was investigated by WB. The amounts of ITIH4 and cit-ITIH4 in synovial fluid (SF) from RA and osteoarthritis (OA) patients were examined by I.P. and W.B. Recombinant ITIH4 and cit-ITIH4 were incubated with sera from healthy volunteers before its chemotactic ability and C5a level were evaluated using Boyden's chamber assay and enzyme-linked immunosorbent assay (ELISA). During peak arthritic phase, ITIH4 and cit-ITIH4 were increased in joints while PAD4 was over-expressed, especially in the infiltrating neutrophils of pGIA mice. Levels of cit-ITIH4 in plasma and joints significantly decreased upon neutrophil depletion. ITIH4 was specifically citrullinated in SF from RA patients compared with OA patients. Native ITIH4 inhibited neutrophilic migration and decreased C5a levels, while cit-ITIH4 increased its migration and C5a levels significantly. Cit-ITIH4 is generated mainly in inflamed joints by neutrophils via PAD4. Citrullination of ITIH4 may change its function to up-regulate neutrophilic migration by activating the complement cascade, exacerbating arthritis.

Efficiency of genomic selection for breeding population design and phenotype prediction in tomato.

Genomic selection (GS), which uses estimated genetic potential based on genome-wide genotype data for a breeding selection, is now widely accepted as an efficient method to improve genetically complex traits. We assessed the potential of GS for increasing soluble solids content and total fruit weight of tomato. A collection of big-fruited F1 varieties was used to construct the GS models, and the progeny from crosses was used to validate the models. The present study includes two experiments: a prediction of a parental combination that generates superior progeny and the prediction of progeny phenotypes. The GS models successfully predicted a better parent even if the phenotypic value did not vary substantially between candidates. The GS models also predicted phenotypes of progeny, although their efficiency varied depending on the parental cross combinations and the selected traits. Although further analyses are required to apply GS in an actual breeding situation, our results indicated that GS is a promising strategy for future tomato breeding design.

Electrophoretic mobility shift scanning using an automated infrared DNA sequencer.

Electrophoretic mobility shift assay (EMSA) is widely used in the study of sequence-specific DNA-binding proteins, including transcription factors and mismatch binding proteins. We have established a non-radioisotope-based protocol for EMSA that features an automated DNA sequencer with an infrared fluorescent dye (IRDye) detection unit. Our modification of the elec- trophoresis unit, which includes cooling the gel plates with a reduced well-to-read length, has made it possible to detect shifted bands within 1 h. Further, we have developed a rapid ligation-based method for generating IRDye-labeled probes with an approximately 60% cost reduction. This method has the advantages of real-time scanning, stability of labeled probes, and better safety associated with nonradioactive methods of detection. Analysis of a promoter from an industrially important filamentous fungus, Aspergillus oryzae, in a prototype experiment revealed that the method we describe has potential for use in systematic scanning and identification of the functionally important elements to which cellular factors bind in a sequence-specific manner.

Differential expression of two BMP antagonists, gremlin and Follistatin, during development of the chick feather bud.

Expression of four BMP antagonist genes, noggin, chordin, gremlin and Follistatin, was examined during chick feather development. Although expression of noggin and chordin was not detected, gremlin and Follistatin were expressed differentially in feather buds. The differential expression patterns of gremlin and Follistatin change dynamically from the nascent inter-feather bud region to the posterior domain of the feather bud.

Globular tail of myosin-V is bound to vamp/synaptobrevin.

VAMP/synaptobrevin is one of a number of v-SNAREs involved in vesicular fusion events in neurons. In a previous report, VAMP was shown to form a complex with synaptophysin and myosin V, a motor protein based on the F-actin, and that myosin V was then released from the complex in a Ca(2+)-dependent manner. Here, we found that VAMP alone is bound to myosin V in a Ca(2+)-independent manner, and determined that the globular tail domain of myosin V is its binding site. The syntaxin-VAMP-myosin V formed in the presence of Ca(2+)/calmodulin (CaM). In the absence of CaM, only syntaxin-VAMP, or VAMP-myosin V complex was formed. Our results suggest that VAMP acts as a myosin V receptor on the vesicles and regulates formation of the complex.

Characterization of powder components of commercial bone cements.

Acrylic bone cements have been used in orthopedic surgery without detailed information on their basic characteristics, especially on their powder components. In this study, the powder components of seven bone cements available on the market in Japan were characterized for morphology, polymer structure and molecular weight, content of residual monomer and benzoyl peroxide (BPO), and thermal properties using scanning electron microscopy, nuclear magnetic resonance spectroscopy, size exclusion chromatography, high performance liquid chromatography, and differential scanning calorimetry, respectively. Considerable differences between the seven bone cements were found in polymer structure and molecular weight, and especially in BPO content and in the morphology of the polymer particles such as shape, size and distribution. It was found that the BPO content was not always in agreement with the value given by the manufacturers on the package.

The mechanism of the neurotransmitter release in growth cones.

The growth cone is considered the precursor of the presynaptic terminal. To elucidate the minimal molecular machinery required for exocytosis, we examined the characteristics of alpha-latrotoxin-induced exocytosis in growth cones. In isolated growth cones (IGC), neurotransmitters were released in a SNARE-dependent manner, but rab3A cycling was blocked. By supplying rabphilin, a rab3A acceptor found in low levels in IGC, the IGC obtained as high an exocytotic efficiency as adult synaptosomes, and the complete GDP-GTP conversion of rab3A occurred on growth cone vesicles (GCV). GCVs bound SNAREs but not NSF or alpha-SNAP; whereas in the rabphilin-supplied IGC, GCVs recruited both NSF and alpha-SNAP, to form the SNARE-NSF-SNAP complex. These results suggest that rab3A cycling is dependent upon the accumulation of rabphilin and is completed later than the SNARE mechanism, and that rabphilin is involved in determining the efficiency of exocytosis by modifying the SNARE mechanism.

An automated system for genome analysis to support microbial whole-genome shotgun sequencing.

We developed a semi-automated genome analysis system called GAMBLER in order to support the current whole-genome sequencing project focusing on alkaliphilic Bacillus halodurans C-125. GAMBLER was designed to reduce the human intervention required and to reduce the complications in annotating thousands of ORFs in the microbial genome. GAMBLER automates three major routines: analyzing assembly results provided by genome assembler software, assigning ORFs, and homology searching. GAMBLER is equipped with an interface for convenience of annotation. All processes and options are manipulatable through a WWW browser that enables scientists to share their genome analysis results without choosing computer platforms.

Halothane facilitates the translocation of GRK-2 and phosphorylation of beta2-adrenergic receptor in rat synaptosomes.

PURPOSE: To examine the effect of halothane on beta2-adrenergic receptor phosphorylation and on G-protein coupled receptor kinase (GRK), responsible for beta2-receptor downregulation. METHODS: Rat forebrain synaptosomes were incubated for 30 min with halothane 1 or 2%. The cytosolic and membrane fractions were separated, and phosphorylation activity of recombinant beta2-adrenergic receptor was quantified autoradiographically using 32P labeled adenosine triphosphate. Phosphorylation activity of a specific GRK-2 substrate, was examined by measuring 32P binding. Subcellular localization of the enzyme was immunologically analyzed by Western blotting. RESULTS: Halothane 2% decreased the phosphorylation activity of the recombinant receptor in the cytosol fraction, regardless of 10 microM isoproterenol (ISP) (P<0.01), which activity in the membrane fraction was increased (P<0.01). Phosphorylation activity of the synthetic peptide decreased in the cytosol obtained from synaptosomes exposed to halothane 2% (P<0.05). In contrast, activity in the membrane increased by exposure to halothane 2% (P<0.01). The concentration of GRK-2 decreased in the cytosol obtained from synaptosomes exposed to halothane 1% or 2% (decreases of 8.3+/-1.2% @ 1%, and 18.0+/-2.1% @ 2%, P<0.05). In the membrane, exposure to halothane 1% or 2% increased the GRK-2 amount dose dependently (22.5+/-3.1% @ 1%, and by 45.7+/-6.1% @ 2%, P<0.01). CONCLUSION: Halothane could facilitate translocation of GRK-2 and possibly promote the downregulation of beta2-adrenergic receptors in the synaptic membrane. The anesthetic action and hemodynamic suppressive action of halothane may be related to this phenomenon.

Differential scanning calorimetric study of acrylic resin powders used in dentistry.

The thermal behavior of eight dental acrylic resin powders was studied using differential scanning calorimetry (DSC). In addition, high performance liquid chromatography was performed to supplement the DSC analysis. The HPLC analysis revealed that the contents of residual monomers and benzoyl peroxide (BPO) in the powders were 0.01-0.97 mass% and 0.25-1.28 mass%, respectively. All the resin powders produced one broad exothermic peak, while a mixture of BPO and PMMA powders generated two peaks. One peak pattern was assigned to the decomposition of BPO included within the polymer particles. The results suggested that BPO was present inside the particles and little BPO was mixed into the resin powders. Moreover, the present study demonstrated a unique useability of DSC in characterizing resin powders.

The effect of immunosuppressive agents (FK-506, rapamycin) on renal P450 systems in rat models.

It is well known that cyclosporin, rapamycin and FK-506 (tacrolimus) are metabolized by the liver microsomal cytochrome P450 enzyme system. Although there have been reports of interaction between these drugs and the renal P450 enzyme system, differences among these immunosuppressants has not been comprehensively demonstrated. We have studied the individual capacities of these immunosuppressants to induce renal microsomal P450 enzymes similar to CYP2B4 and CYP4A2 by examining renal function in treated rats, and have correlated the results by means of biochemical, immunological and immunohistochemical assays of renal P450 enzymes. Cyclosporin caused impairment of renal function with an increase in renal-specific P450 content, but FK-506 and rapamycin did not. Laurate omega- and (omega-1)-hydroxylase activity increased in rats treated with rapamycin but decreased in those treated with FK-506. Prostaglandin A1 (PGA1) omega-hydroxylase activity increased in rats treated with FK-506 but was reduced by treatment with cyclosporin. Aminopyrine N-demethylase activity increased in rats treated with cyclosporin or FK-506, but not in those treated with rapamycin. Western-blot analysis revealed significant induction of P450, (similar to CYP2B4 of the rabbit P450 isozyme) in kidneys from rats treated with cyclosporin but not in those from rats receiving FK-506 or rapamycin. Histochemical studies clearly demonstrated a form of P450 such as CYP4A2 in the proximal tubules of rats treated with cyclosporin, but not in those of rats treated with FK-506 or rapamycin. These results show that although cyclosporin has a strong effect on renal P450 systems and induces such a system in kidney cortex (microsomal P450), FK-506 and rapamycin have no substantial effect on the induction of renal P450. These findings might clarify the nephrotoxicity induced by these immunosuppressive drugs.

An intrinsic DNA curvature found in the cyanobacterium Microcystis aeruginosa K-81 affects the promoter activity of rpoD1 encoding a principal sigma factor.

The rpoD1 gene in the unicellular cyanobacterium Microcystis aeruginosa K-81 encodes a principal sigma factor of RNA polymerase and is transcribed under light and dark conditions to produce multiple monocistronic transcripts. In the 5'-upstream region from rpoD1 Promoter 2, which has a sequence of Escherichia coli type, we found a sequence-directed DNA curvature with an AT-rich sequence. Insertions of 2 to 21 base pairs introduced into the curved center changed a gross geometry of the original curved DNA structure. The rpoD1 promoter activities assayed in vivo by using transcriptional lacZ fusions were correlated with the change in the gross geometry in not only a cyanobacterium but also E. coli. In addition, RNA polymerase binding to the rpoD1 promoter region and the efficiency of the mRNA synthesis from the rpoD1 Promoter 2 were also affected in vitro by the change in the geometry. These results suggest that the tertiary structure of the curved DNA is important for the rpoD1 transcription. The deletion of the center region of the curvature resulted in a considerable reduction of the transcription from Promoter 2 in the cyanobacterium. This report demonstrates that a curved DNA plays a significant role in transcription in cyanobacteria, and that this functional curvature is located in the 5'-upstream region from the rpoD gene, which encodes a principal sigma factor in eubacteria.

Purely laparoscopic pylorus-preserving gastrectomy with extraperigastric lymphadenectomy for early gastric cancer: a case and technical report.

For the purpose of prevention of postgastrectomy syndrome and a less invasive and yet curative oncological resection, a purely laparoscopic pylorus-preserving gastrectomy with extraperigastric lymphadenectomy was performed for a patient with early gastric cancer located in the middle third of the stomach. The patient's postoperative course was uneventful. During his postoperative recovery, the patient experienced very little pain and used analgesic medication only one time. This operation appeared to be oncologically adequate. As of the seventh postoperative month, the patient never experienced dumping syndrome or alkaline reflux gastritis. This procedure is technically feasible and an excellent option because of its reduced surgical invasiveness and better postoperative quality of life.

Cloning of cDNA for a cell wall-bound acid invertase from tomato (Lycopersicon esculentum) and expression of soluble and cell wall-bound invertases in plants and wounded leaves of L. esculentum and L. peruvianum.

A cDNA, Wiv-1, for an isozyme of acid invertase (EC 3.2.1.26) was cloned from wounded leaves of tomato (Lycopersicon esculentum). The encoded protein had a basic isoelectric point and strong similarity to the amino acid sequences of plant cell wall-bound invertases. The conserved sequence WECPD that is found in all plant cell wall-bound invertases was also found in the deduced protein. These results suggested that Wiv-1 encoded a cell wall-bound acid invertase of tomato. Wounding increased the levels of mRNAs for soluble and cell wall-bound invertases and the activities of these invertases in leaves of L. esculentum and of a related species, L. peruvianum. The induction of Aiv-1 mRNA for the soluble enzyme in wounded leaves was not very strong, while that of Wiv-1 mRNA for the wall-bound enzyme was prominent. The level of Aiv-1 mRNA reached a maximum 48 h after wounding while that of Wiv-1 mRNA continued to rise for up to 96 h. These findings suggested that the genes for the two isozymes responded independently to wounding. The levels in various organs of Aiv-1 and Wiv-1 mRNAs were higher in L. esculentum than in L. peruvianum. Possible roles of cell wall-bound acid invertase in wound response and in developing plant are discussed.