Incidence and risk factors for ocular GVHD after allogeneic hematopoietic stem cell transplantation.

We analyzed the incidence and risk factors for ocular GVHD in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) in Korea. In this retrospective, noncomparative, observational study, 635 subjects were included who had at least 2 years of follow-up ophthalmological examinations after allo-HSCT from 2009 to 2012 at Seoul St Mary's Hospital, Seoul, Korea. The mean duration between allo-HSCT and onset of ocular GVHD was 225.5±194.3 days. The adjusted incidence for acute ocular GVHD was 1.33% and that for chronic GVHD was 33.33%. In the multivariate analysis, preexisting diabetes mellitus (odds ratio (OR): 4.22, 95% confidence interval (CI): 1.66-10.72), repeated allo-HSCT (OR: 29.10, 95% CI: 1.02-8.28) and the number of organs that chronically developed GVHD by stage I (OR: 14.63, 95% CI: 9.81-21.84) increased risk of ocular GVHD. Careful monitoring of ocular GVHD is needed in patients with chronic GVHD in multiple organs and preexisting diabetes.

Simulation and sensitivity analysis of phosphorylation of EGFR signal transduction pathway in PC12 cell model.

The epidermal growth factor receptor (EGFR) signalling pathway is a complex signalling process with a wide network of interactions. The activation of the mitogen-activated protein kinases (MAPKs) cascade by this activated EGFR has been well studied. MAPKs form a highly integrated network, which is essential for certain specialised cell functions. This paper presents a kinetic model for the MAPK pathway downstream of the EGFR using a biochemical simulator. The model includes 30 signalling events and 29 signalling molecules. The time course data were examined for the activation of each signalling component. The simulation provides a large volume of data, by monitoring the kinetics of the signalling components, which were compared experimentally using the PC12 cell line. The kinetic model corresponded well with the experimental results observed in the EGFR induced activation of proteins. An examination of the kinetic analysis of the multiple signalling events provides a quantitative framework for representing the EGFR signalling network.

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection.

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.

Measurement of testosterone and pregnenolone in nails using gas chromatography-mass spectrometry.

An efficient method for the determination of testosterone and pregnenolone in human nails using gas chromatography-mass spectrometry (GC-MS) with d3-testosterone as an internal standard is described. The method involves alkaline digestion and liquid-liquid extraction, with subsequent conversion to mixed pentafluoropenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected-ion monitoring (SIM) mode. The limit of detection (LOD) and limit of quantification (LOQ) were lowered to 0.1 and 0.2 pg/g, respectively, when 100 mg of nail-clippings were used. The mean recoveries of testosterone and pregnenolone were 89.8 and 86.7%, respectively, while good overall precision (% C.V.; 4.5-9.5) and accuracy (% bias; 3.9-8.4) were demonstrated. Linearity as a correlation coefficient was 0.9913 (testosterone) and 0.9965 (pregnenolone). When applied to fingernail and toenail samples from seven healthy men and nine healthy women, testosterone and pregnenolone were positively detected in the concentration range of 0.24-5.80 ng/g. The levels of two steroids studied in the nails were found to be higher in the male subjects than in the female subjects, and except for the toenails of the females, the levels of testosterone were higher than those of pregnenolone.

Phlegmonous enteritis in a patient with congestive heart failure and colon cancer.

Phlegmonous enteritis is a rare infective inflammatory disease of the intestine, predominantly involving the submucosal layer. It is difficult to diagnose and often fatal. Its association with alcoholism and various liver diseases, although rarely reported, is well documented. We report a case of phlegmonous enteritis in a male patient with congestive heart failure and colon cancer, and describe the ultrasonographic and CT findings.

Huge biliary cystadenoma mimicking cholecystic lymphangioma in subhepatic space.

Biliary cystadenoma is a rare cystic neoplasm and constitutes only 5% of all intrahepatic cysts of biliary origin. We report a case of a 44-year-old woman with huge biliary cystadenoma in the subhepatic space, mimicking a cholecystic lymphangioma. Findings of various imaging modalities including reconstructed CT image are presented and correlated with surgical and pathologic findings.

Characterization of epidermal growth factor receptor function in lysophosphatidic acid signaling in PC12 cells.

Lysophosphatidic acid (LPA) is a lipid metabolite that induces the activation of mitogen-activated protein kinase (MAPK) through binding to the G protein-coupled receptor in a number of cell lines and cultures. Recent studies have revealed that LPA is able to rapidly induce the phosphorylation of MAPK through an epidermal growth factor (EGF) receptor-dependent pathway. We investigated the role of the EGF receptor in the signaling pathway initiated by LPA stimulation in nerve growth factor (NGF)-responsive PC12 cells well known to transiently retract their own neurites upon LPA stimulation. LPA-stimulated MAPK signaling was suppressed by the selective EGF receptor inhibitor and in the dominant negative mutant EGF receptor cell line. As in the EGF signaling pathway, the complex of EGF receptor with adapter proteins Shc and Sos was formed in response to LPA stimulation, suggesting there is an intracellular mechanism for transactivation. A neurite retraction assay was also performed to examine the role of the EGF receptor in PC12 cell differentiation, which related to the involvement of LPA-induced neurite retraction. These results suggest that the receptor tyrosine kinase can be activated in a ligand-independent manner through intracellular crosstalk between the signaling pathways.

Proliferation not apoptosis as a prognostic indicator in retinoblastoma.

The balance between proliferation and cell death is the major determinant of tumour growth. We analysed the proliferative and apoptotic indices (PI and AI, respectively) of 33 children with retinoblastoma. PI and AI were assessed by immunohistochemistry for Ki-67 antigen and TUNEL staining, respectively. The mean PI was 21.0+/-21.1%, and higher PI was associated with more advanced tumour stage (P<0.0001) and poor clinical outcome (P<0.05). Patients in whom amplified N-myc oncogene was found (n=6) determined by the multiplex polymerase chain reaction tended to have a higher PI (37.6+/-27.2%) than those without amplified N-myc (n=27; PI=17.3+/-18.1). A PI value of over 40% was clearly associated with an unfavourable prognosis. The AI, however, did not correlate with any of the other variables analysed. The findings suggest that proliferation, but not apoptosis, is of critical significance in retinoblastoma biology. PI, as determined by the Ki-67 antigen labelling index, seems to be a relevant histopathological parameter that can predict the clinical outcome of retinoblastoma.

Application of capillary electrophoresis to determination of enzyme activity and other properties of phosphatidylinositol-specific phospholipase C.

This paper describes a method for monitoring enzymatic activity using micellar electrokinetic chromatography (MEKC). MEKC is used to analyze the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) with convenience and precision. PI specific PLC enzyme converts phosphatidylinositol (PI) to diacylglycerol (DAG) and inositol 1,2-cyclic phosphate (IP). The assay system developed is based on monitoring both the breakdown of substrate and the formation of products simultaneously. To obtain the best separation for the substrate PI and product DAG, we investigated changes in concentration of electrolyte buffer and SDS as well as in pH of the electrolyte buffer. Since the structures of the substrate and products are different, the reaction could be monitored easily by MEKC mostly based on hydrophobicity. Under the separation conditions developed, we investigated the enzymatic activity of PI-PLC depending on the concentrations of phosphatidylinositol, various divalent cations and the reaction temperature. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to confirm the molecular masses of the substrate PI and products DAG and IP. The results of this study show that capillary electrophoresis can be widely applied to analyze and characterize many other enzymes since capillary electrophoresis has several advantages as follows, simplicity, speed, no need for radiolabelled substrate, small sample volumes and sufficient accuracy.

Separation of gangliosides using cyclodextrin in capillary zone electrophoresis.

Gangliosides are glycosphingolipids containing sialic acid. These glycolipids have been suggested to play important roles in biological processes such as cell growth, differentiation and malignant transformation. Based on these proposed biological functions, gangliosides can be used as diagnostic tools and therapeutics for various human diseases. In this study, capillary zone electrophoresis (CZE) was used to determine the major gangliosides GM1, GD1a, GD1b and GT1b in mammalian brains, in addition to GM3 and Lac. Enhancement of selectivity and efficiency of separation was obtained by using 50 mM borate-phosphate buffer containing 16.5 mM alpha-cyclodextrin (alpha-CD). Under this condition, several forms of gangliosides were successfully separated from extracts of deer antler, apricot seed and rat brain. The results demonstrate that the CD-modified CZE is a useful method for detecting glycolipids from various biological matrices.

Genotoxicity of beryllium, gallium and antimony in short-term assays.

The genotoxicity of beryllium, gallium and antimony compounds was studied with the rec, Salmonella mutagenicity and SCE assays. In the rec assay, all the salts of the metals, BeCl2, Be(NO3)2, GaCl3, Ga(NO3)3, SbCl3, SbCl5, and an oxide, Sb2O3, had DNA-damaging activity. None of the compounds was mutagenic to Salmonella. In the SCE assays using V79 cells, 2 antimony(III) compounds, SbCl3 and Sb2O3, and 2 beryllium compounds, BeCl2 and Be(NO3)2, induced SCEs significantly. Sb2O3, slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses.

Primary structure of bovine adrenal phenylethanolamine N-methyltransferase.

Bovine adrenal medullary phenylethanolamine N-methyltransferase (E.C. 2.1.1.28) was sequenced to determine if primary structure or post-translational processing accounts for the charged isozymes. A blocked NH2-terminus precluded amino terminal sequencing. Therefore, cyanogen bromide and tryptic peptide fragments were isolated and subjected to gas phase/gas-liquid phase sequencing. Primary structure was identified for 45% of the protein. At least two polypeptide chains exist with alternative amino acids representing conservative changes or single-base changes in amino acid codons by comparison to the cDNA sequence for the enzyme.