• Journal of Genetic Medicine
• /
• v.4 no.1
• /
• pp.45-52
• /
• 2007

Purpose : A simple, rapid, and highly sensitive analytical method for Gb3 in plasma was developed without labor-ex tensive pre-treatment by electrospray ionization MS/ MS (ESI-MS/MS). Measurement of globotriaosy lceramide (Gb3, ceramide trihex oside) in plasma has clinical importance for monitoring after enzyme replacement therapy in Fabry disease patients. The disease is an X-linked lipid storage disorder that results from a deficiency of the enzyme ${\alpha}$-galactosidase A (${\alpha}$-Gal A). The lack of ${\alpha}$-Gal A causes an intracellular accumulation of glycosphingolipids, mainly Gb3. Methods : Only simple 50-fold dilution of plasma is necessary for the extraction and isolation of Gb3 in plasma. Gb3 in diluted plasma was dissolved in dioxane containing C17:0 Gb3 as an internal standard. After centrifugation it was directly injected and analyzed through guard column by in combination with multiple reaction monitoring mode of ESI-MS/MS. Results : Eight isoforms of Gb3 were completely resolved from plasma matrix. C16:0 Gb3 occupied 50% of total Gb3 as a major component in plasma. Linear relationship for Gb3 isoforms w as found in the range of 0.001-1.0 ${\mu}g$/mL. The limit of detection (S/N=3) was 0.001 ${\mu}g$/mL and limit of quantification was 0.01 ${\mu}g$/mL for C16:0 Gb3 with acceptable precision and accuracy. Correlation coefficient of calibration curves for 8 Gb3 isoforms ranged from 0.9678 to 0.9982. Conclusion : This quantitative method developed could be useful for rapid and sensitive 1st line Fabry disease screening, monitoring and/or diagnostic tool for Fabry disease.

• Journal of Plant Biotechnology
• /
• v.2 no.3
• /
• pp.115-121
• /
• 2000

A cDNA clone encoding chloroplast triosephosphate isomerase (TPI-cp) was isolated from strawberry fruit cDNA library. Sequence analyses indicated that the cDNA contains an open reading frame of 314 amino acids (33.5 kDa) composed of a transit peptide (59 amino acids) in amino terminal region and mature protein (255 amino acids). The existence of transit peptide in the deduced amino acid sequence implies that it encodes a chloroplast isoform. The protein sequence is more similar to other plant chloroplast isoforms than cytosolic isoforms. RNA blot analysis indicated that its expression is ubiquitous in examined five tissues, flowers, leaves, petioles, roots and fruits, and shows differential pattern according to fruit ripening. Genomic DNA blot analysis showed that TPI-cp is encoded by multiple genes in strawberry. Through sequence comparison and phylogenetic tree construction, TPI-cp is distinctively grouped into dicot and chloroplast isoforms.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.3
• /
• pp.472-476
• /
• 2008

Two isoforms of human CD99 have been identified, but only heterotypic interaction between the isomers was recently demonstrated. In this study, we performed bimolecular fluorescence complementation analysis to further characterize the interaction in vivo. Upon transiently transfecting plasmids expressing either of the two isoforms fused with yellow fluorescent protein (YFP) fragments, all the YFP-tagged CD99 molecules were properly localized on cell surfaces, and formed fluorescent dimers. Interestingly, however, unlike the previous report, the homodimers formed as efficiently as the heterodimer via their extracellular domains, implying its distinct regulatory role through modulating the complex profile.

• Animal cells and systems
• /
• v.13 no.3
• /
• pp.283-288
• /
• 2009

The ubiquitous plasma membrane $Na^+/H^+$ exchanger 1 (NHE1) is rapidly activated in response to various extracellular stimuli and maintains normal cytoplasmic pH. Yeast two-hybrid screening was used in order to identify proteins interacting with NHE1 using its cytoplasmic domain as a bait from HeLa cDNA library. One of the interacting cDNA clones was human Lactate dehydrogenase B (LDHB). In vitro translated LDHB was pulled down together with GST-NHE1.cd protein in the GST pull down assay, confirming the interaction in vitro. LDHB antibody immunoprecipitated endogenous LDHB together with NHE1 from H9c2 cells, validating cellular interaction between NHE1 and LDHB. Subsequent analysis revealed that the overexpression of LDHB increased intracellular PH, implying opening of the NHE1 transporter. Moreover, overexpression of LDHB activated caspase 3 and induced cell death, consistent with the expected phenotype of hyper-activation of NHE1. Collectively, our data indicate that LDHB modulates NHE1 activity via physical interaction.

• BMB Reports
• /
• v.36 no.1
• /
• pp.49-59
• /
• 2003

Platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. The vital functions of PDGFs for angiogenesis, as well as development of kidney, brain, cardiovascular system and pulmonary alveoli during embryogenesis, have been well demonstrated by gene knock-out approaches. Clinical studies reveal that aberrant expression of PDGF and its receptor is often associated with a variety of disorders including atherosclerosis, fibroproliferative diseases of lungs, kidneys and joints, and neoplasia. PDGF contributes to cancer development and progression by both autocrine and paracrine signaling mechanisms. In this review article, important features of the PDGF isoforms and their cell surface receptor subunits are discussed, with regards to signal transduction, PDGF-isoform specific cellular response, and involvement in angiogenesis, and tumorstromal interactions.

• Proceedings of the Korean Society of Plant Biotechnology Conference
• /
• 2005.04a
• /
• pp.62-62
• /
• 2005

• Journal of Food Hygiene and Safety
• /
• v.14 no.4
• /
• pp.415-421
• /
• 1999

The flavin-containing monooxygenases (FMOs) (EC 1.14. 13.8) are NADPH-dependent flavoenzymes that catalyze oxidation of soft nucleophilic heteroatom centers in a range of structurally diverse compounds including foods, drugs, pesticides, and other xenobiotics. In humans, FMOl appears to be the predominant form expressed in human fetal liver. cDNA-expressed human FMO and human liver microsomal FMO have been observed to N- and S-oxy-genate nucleophilic nitrogen- and sulfur-containing drugs and chemicals, respectively. In the present study, FMOl can be expressed in the baculovirus expression vector system at level of 2.68 nmol FMOl/mg of membrane protein. This isoform was examined for its capacity to metabolize methimazole to its S-oxide using thiocholine assay. Kinetic studies of its S-oxide by recombinant human FMO1 result in Km of 7.66 $\mu$M and Vmax of 17.79 nmol/min/mg protein.

• Proceedings of the PSK Conference
• /
• 2002.10a
• /
• pp.248.3-249
• /
• 2002

Nitric oxide (NO) has been shown to play an important role in the regulation of vascular tone. platelet function. neurotransmission. and immune function. NO is synthesized from the L-arginine by NO synthase (NOS). Three distinct isoforms of NOS have been identified: calcium/calmodulin-dependent endothelial (eNOS) and neuronal (nNOS) isoforms which are constitutive and produce small quantities of NO, and an inducible isoform (iNOS) which is markedly induced in response to lipopolysaccharide (LPS) or inflammatory cytokines. (omitted)

• Journal of Ecology and Environment
• /
• v.29 no.2
• /
• pp.157-163
• /
• 2006

Growth of G. max treated with $NO_3^-$-N was decreased by high NaCl treatments, but $NH_4NO_3$-fed plants showed good growth with enhanced activity of antioxidative enzymes (SOD and APX). Especially, activity of APX was higher in 5 mM $NH_4NO_3$-fed plants than other types of N-supplied plants throughout the stress period. Higher SOD activity under salt stress was accompanied by increase in APX activity in 5 mM $NH_4NO_3$-fed plants. Similarly, application of calcium confirmed somewhat positive effects on growth. Salt-treated soybean plants showed the best growth response with the increase of SOD and APX activity at an additional 5 mM calcium treatment. Especially, the increase of SOD activity through the strengthened CuZn-SOD isoform was remarkable.

• Biomedical Science Letters
• /
• v.16 no.4
• /
• pp.207-212
• /
• 2010

Archivillin, a muscle-specific isoform of supervillin, is a component of the costameric cytoskeleton of muscle cells. The purpose of this study was to determine which protein in the skeletal muscle collaborates with archvillin C-terminus. For this purpose, a yeast two-hybrid screening of human skeletal muscle cDNA library was performed using the C-terminal region of archvillin as bait. This study shows that seven human skeletal muscle proteins, namely, nebulin, xeplin, archvillin, GAPDH, TOX4, PITRM1, and YME1L1 interact with archvillin C-terminus. Especially, xeplin is a newly discovered protein interacts with archvillin C-terminus. These results indicate that archvillin C-terminus acts as a bridge between nebulin and xeplin at costameres. Archvillin C-terminal region interacts with nebulin C-terminal region at Z-discs and interacts with xeplin at the vicinity of sarcolemma. I propose that these interactions may contribute to formation of costameric structure and muscle contraction.