Kyu Hwan Park

Identification of trimethylation at C-terminal lysine of pilin in the cyanobacterium Synechocystis PCC 6803.

Various post-translational modifications (PTMs) of pilin in Synechocystis sp. PCC 6803 have been proposed. In this study, we investigated previously unidentified PTMs of pilin by mass spectrometry (MS). MALDI-TOF MS and TOF/TOF MS showed that the molecular mass of the C-terminal lysine of pilin was increased by 42Da, which could represent acetylation (ΔM=42.0470) or trimethylation (ΔM=42.0106). To discriminate between these isobaric modifications, the molecular mass of the C-terminal tryptic peptide was measured using 15T Fourier transform ion cyclotron resonance (FT-ICR) MS. The high magnetic field FT-ICR provided sub-ppm mass accuracy, revealing that the C-terminal lysine was modified by trimethylation. We could also detect the existence of mono- and di-methylation of the C-terminal lysine. Cells expressing a pilin point mutant with glutamine replacing the C-terminal lysine showed dramatically reduced motility and short pili. These findings suggest that trimethylation of pilin at the C-terminal lysine may be essential for the biogenesis of functional pili.

Analysis of cancer cell lipids using matrix‐assisted laser desorption/ionization 15‐T Fourier transform ion cyclotron resonance mass spectrometry

A combination of methodologies using the extremely high mass accuracy and resolution of 15-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) was introduced for the identification of intact cancer cell phospholipids. Lipids from a malignant glioma cell line were initially analyzed at a resolution of >200,000 and identified by setting the mass tolerance to ±1 mDa using matrix-assisted laser desorption/ionization (MALDI) 15-T FT-ICR MS in positive ion mode. In most cases, a database search of potential lipid candidates using the exact masses of the lipids yielded only one possible chemical composition. Extremely high mass accuracy (<0.1 ppm) was then attained by using previously identified lipids as internal standards. This, combined with an extremely high resolution (>800,000), yielded well-resolved isotopic fine structures allowing for the identification of lipids by MALDI 15-T FT-ICR MS without using tandem mass spectrometric (MS/MS) analysis. Using this method, a total of 38 unique lipids were successfully identified.

Effect of repeated seizure experiences on tyrosine hydroxylase immunoreactivities in the brain of genetically epilepsy-prone rats

The genetically epilepsy-prone rat (GEPR) is a model of generalized tonic/clonic epilepsy, and has functional noradrenergic deficiencies that act as partial determinants for the seizure predisposition and expression. The present study investigated the effect of repeated seizure experiences by acoustic stimulation (110 dB, 10 times) on the immunoreactivities of tyrosine hydroxylase (TH), a rate-determining enzyme in the synthesis of norepinephrine, in brain regions of GEPRs. TH immunoreactivity in locus coeruleus, the major noradrenergic nucleus in brain, was lower in GEPRs than control Sprague-Dawley rats. It was also decreased in several regions including inferior colliculus of GEPRs. Repeated experiences of audiogenic seizures further decreased TH immunoreactivities in locus coeruleus and inferior colliculus of GEPRs. The results from the present study suggest that the lower immunoreactivities of TH in locus coeruleus and inferior colliculus contribute, at least in part, to the noradrenergic deficits in GEPRs, and repeated seizure experiences further intensified these noradrenergic deficits, which may be related to the altered seizure expression by repetitive audiogenic seizure in GEPRs.

INHALED ATP CAUSES MUCIN RELEASE FROM GOBLET CELLS OF INTACT RATS

Secretion of mucins from airway epithelial cells has been studied almost exclusively using in vitro cell culture systems. Our understanding of in vivo secretion is greatly limited due to the unavailability of both suitable model systems and adequate assays. It has been reported that ATP induces mucin release from the cultured primary tracheal surface epithelial cell, but there is no clear demonstration of the effect of ATP on mucin release in vivo, which is important to understand the mechanism of mucin release in vivo and also to devise means for regulation of mucin release. The objective of this experiment was to see if inhaled ATP could stimulate airway mucin release in intact rats using both enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. The results were: (1) a new monoclonal antibody (mAbRT03) developed against purified rat mucins specifically recognized high-molecular-mass mucins; (2) ELISA results with conventional gel-filtration assay results are virtually superimposable; (3) inhalation of ATP in intact rats resulted in a dose-independent increase in the amount of mucins in the tracheal lavage fluid with a concomitant decrease in the number of mucin-positive cells in the trachea. We conclude that extracellular ATP can stimulate mucin release from the airway in vivo, and the present rat inhalation system combined with ELISA of the airway secretions should serve a useful model for studying the pharmacology of airway mucin secretion in vivo.

Clarification of a peak at m/z 1634 from tryptically digested cytochrome c.

A peptide peak at m/z 1634 in the mass spectrum of tryptically digested cytochrome c has been ambiguously assigned to either a peptide IFVQKCAQCHTVEK or a peptide CAQCHTVEK combined with a heme group (CAQCHTVEK + heme (Fe(III))). A comprehensive investigation was performed to clearly identify the origin of the peak. Tryptic digests of cytochrome c were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography-tandem MS (LC-MS/MS), LC-ultraviolet (LC-UV), and MALDI Fourier transform-ion cyclotron resonance (FT-ICR) MS. The use of instruments with extremely high mass accuracy revealed the mass difference between the IFVQKCAQCHTVEK and the (CAQCHTVEK + heme (Fe(III))) ions. Fragmentation of the peptide associated with the unknown peak yielded a heme ion and other fragment ions originating from a (CAQCHTVEK + heme (Fe(III))) ion. Furthermore, an absorption peak at 395 nm confirmed the presence of a heme group in the unknown peptide. High mass accuracy analyses of MS and MS/MS spectra, in addition to three-dimensional UV contour mapping, showed that the peak at m/z 1634 is due to a (CAQCHTVEK + heme (Fe(III))) ion and not from protonated IFVQKCAQCHTVEK.

Metabolic changes in serum steroids induced by total-body irradiation of female C57B/6 mice

The short- and long-term effects of a single exposure to gamma radiation on steroid metabolism were investigated in mice. Gas chromatography-mass spectrometry was used to generate quantitative profiles of serum steroid levels in mice that had undergone total-body irradiation (TBI) at doses of 0Gy, 1Gy, and 4Gy. Following TBI, serum samples were collected at the pre-dose time point and 1, 3, 6, and 9 months after TBI. Serum levels of progestins, progesterone, 5β-DHP, 5α-DHP, and 20α-DHP showed a significant down-regulation following short-term exposure to 4Gy, with the exception of 20α-DHP, which was significantly decreased at each of the time points measured. The corticosteroids 5α-THDOC and 5α-DHB were significantly elevated at each of the time points measured after exposure to either 1 or 4Gy. Among the sterols, 24S-OH-cholestoerol showed a dose-related elevation after irradiation that reached significance in the high dose group at the 6- and 9-month time points.

Cross‐species reactivity of a monoclonal antibody against glutathione S‐transferase fusion protein of human β2‐adrenergic receptor

The purpose of the present study was to produce and characterize a monoclonal antibody against human β2‐adrenergic receptor. Male BALB/c mice were immunized with glutathione S‐transferase (GST) fusion protein of the C‐terminal portion of the human β2‐adrenergic receptor which was expressed in E.Coli. The immunized splenocytes were fused with myeloma SP2/0‐Ag14 cells and the resulting monoclonal antibody was named as mAbβC02. The monoclonal antibody βC02 was determined as IgM subtype and then purified by anti‐mouse IgM‐agarose affinity chromatography. The results of ELISA, Western blot, and immunocytochemistry showed that mAbβC02 recognized human β2‐adrenergic receptor in the β2‐adrenergic receptor‐GST fusion protein and human epidermoid carcinoma cell line A431 with highly specific immunoreactivity. In addition, mAbβC02 showed cross‐species reactivity against β‐adrenergic receptor of hamster lung and rat brain as revealed by Western blot and immunohistochemistry. The monoclonal antibody βC02 may provide useful tools for the study of the β‐adrenergic receptor of human and other species including rats.

P003: Current status of infection control practice for prevent of central venous catheter-associated bloodstream infection in Korea

There are evidence-based guidelines for the prevention of central line-associated bloodstream infections (CLA-BSI), but the current status of these practices in intensive care units (ICU) of Korea is unknown.