Biomedical subjects

Joon-Yong Chung

Publications and source records attributed to Joon-Yong Chung.

15 recordsLinked to original sources

PubMed2006

Functional identification of a protein inhibitor of neuronal nitric oxide synthase of Taenia solium metacestode.

The protein inhibitor of neuronal nitric oxide synthase (PIN) performs critical functions in several biological processes including inhibition of neuronal nitric oxide synthase (nNOS) activity, intracellular trafficking of proteins and cellular maturation. In this study, we isolated a gene that putatively encoded a PIN homologue in the Taenia solium metacestode (TsM), a causative agent for neurocysticercosis (NC). A full-length cDNA of 452-bp in length, designated TsMPIN, was found to encode an open reading frame (ORF) of 103 amino acids with a predicted molecular weight of 11.3kDa. This single copy gene possessed an intervening short intron (74bp-long) within its ORF region. The deduced amino acid sequence revealed a substantial degree of sequence identity with the PINs and the dynein light-chains isolated from other organisms (63-81%). TsMPIN ectopically expressed in neuroblastoma N1E115 cells effectively inhibited dimerization of nNOS upon stimulation. The recombinant TsMPIN also negatively regulated the dimerization of recombinant nNOS, which was attenuated significantly by the TsMPIN-specific antibody. TsMPIN was primarily localized in the lining cells of the trabecules and the muscles surrounding the scolex, and was sparsely within the cytosol of the bladder wall. We also identified TsM nNOS-immunoreactive protein by both NADPH-diaphorase histochemical staining, and immunohistochemical localization and immunoprecipitation with antibodies specific to nNOS N-terminus. These two functionally related proteins showed a co-localized expression pattern. Our results strongly suggest that the production of NO in the TsM might be tightly regulated through the nNOS-TsMPIN feedback system to maintain physiological homeostasis in the parasite.

Amino Acid Sequence↗

PubMed2006

Tissue microarrays as a platform for proteomic investigation.

Tissue microarrays have become an essential tool in translational pathology. They are used to confirm results from other experimental platforms, such as expression microarrays, as well as a primary tool to explore the expression profile of proteins by immunohistochemical analysis. Tissue microarrays are routinely used molecular epidemiology, drug development and determining the diagnostic, prognostic and predictive value of new biomarkers. By applying traditional protein based assays, as well as novel assays to the platform, tissue microarrays have gained a new utility as a proteomic tool for both basic science as well as clinical investigation. This article will explore the new approaches that are being applied to tissue microarrays to, characterize the human proteome, and new technologies that allow tissue microarrays to function as a protein array.

Animals↗

PubMed2006

Transfer and multiplex immunoblotting of a paraffin embedded tissue.

As we transition from genomics to the challenges of the functional proteome, new tools to explore the expression of proteins within tissue are essential. We have developed a method of transferring proteins from a formalin fixed, paraffin embedded tissues section to a stack of membranes which is then probed with antibodies for detection of individual epitopes. This method converts a traditional tissue section into a multiplex platform for expression profiling. A single tissue section can be transferred to up to ten membranes, each of which is probed with different antibodies, and detected with fluorescent secondary antibodies, and quantified by a microarray scanner. Total protein can be determined on each membrane, hence each antibody has its own normalization. This method works with phospho-specific antibodies as well as antibodies that do not readily work well with paraffin embedded tissue. This novel technique enables archival paraffin embedded tissue to be molecularly profiled in a rapid and quantifiable manner, and reduces the tissue microarray to a form of protein array. This method is a new tool for exploration of the vast archive of formalin fixed, paraffin embedded tissue, as well as a tool for translational medicine.

Antibodies, Phospho-Specific↗

PubMed2006

Optimization of recovery of RNA from formalin-fixed, paraffin-embedded tissue.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for application of diagnostic assays on tissue after microscopic examination. Not only is there a substantial archive of tissue available, but FFPE tissue remains the best method of preparation for microscopic examination in a routine clinical environment. Molecular assays, especially reverse transcription and polymerase chain reaction and expression array-based assays, offer significant potential as diagnostic, prognostic, and predictive tools, but require high quality RNA. Herein, we have optimized a reliable RNA extraction method for FFPE tissue. It is based on deparaffinization at high temperature coupled with a 3-day lysis at 65 degrees C. The average total RNA yield is 4.5 to 5.5 pg per 1 microm of archival FFPE tissue, and 260/280 ratios are between 1.80 and 1.95. The extracted RNA has a modal fragment length between 100 and 200 nt by the Bioanalyzer analysis. Although modal lengths of RNA fragments were shorter, reverse transcription and polymerase chain reaction was able to amplify amplicons in range of 300 bp. Pretreatment with RNA, later followed by formalin fixation, did not result in improving the RNA quality, but did improve RNA yield. Our method improves the utility of FFPE tissue for molecular profiling studies.

Animals↗

PubMed2006

A multiplex tissue immunoblotting assay for proteomic profiling: a pilot study of the normal to tumor transition of esophageal squamous cell carcinoma.

Esophageal cancer remains a highly lethal malignancy for which the genetic and proteomic events are poorly understood. Studies have reported dysregulated proteins in esophageal carcinoma; however, the magnitude of these changes remains largely uncharacterized. Little is known about alterations early in the neoplastic pathway. Using multiplex tissue immunoblotting, we quantified the expression of seven proteins in esophageal carcinogenesis. Regions of normal, dysplasia, and invasive carcinoma of the squamous esophagus in six patients were characterized. Pan-cytokeratin (CK) was essentially unchanged across the transition (0.96 in dysplasia and 0.69 in tumor). Expression levels of annexin 1, CK-4, and CK-14 were all decreased in dysplasia and tumor compared with normal (reference, 1.00): annexin 1, 0.30 in dysplasia and 0.15 in tumor; CK-4, 0.20 in dysplasia and 0.16 in tumor; and CK-14, 0.54 in dysplasia and 0.40 in tumor. Expression of two proteins was increased in dysplasia and tumor versus normal: cyclooxygenase-2, 1.35 in dysplasia and 2.32 in tumor and p53, 1.29 in dysplasia and 2.37 in tumor. Secreted protein, acidic and rich in cysteine, which is expressed in the adjacent stroma, was 1.56-fold higher in stroma underlying dysplasia and 6.20-fold increased in dysplastic stroma surrounding invasive tumor. These findings suggest that changes in protein expression can be detected during the transition to dysplasia and may be useful biomarkers.

Biomarkers, Tumor↗

PubMed2005

Subunit composition of ATP-sensitive potassium channels in mitochondria of rat hearts.

Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a pivotal role in early and late ischemic preconditioning, but the subunit composition of mitoKATP channels remains unclear. In this study, we investigated the subunit composition of mitoKATP channels in rat hearts using confocal microscopy, immunofluorescence, and Western blot analysis. The green fluorescent probe glibenclamide-BODIPY was colocalized with the red fluorescent mitochondrial marker MitroTracker Red in isolated ventricular myocytes and in ventricular myocyte mitochondria, indicating the presence of sulfonylurea receptors (SURs) in the mitochondria. Anti-Kir6.1, anti-Kir6.2, and anti-SUR2 immunofluorescence was colocalized with that of MitoTracker Red in isolated mitochondria, suggesting that Kir6.1, Kir6.2, and SUR2 subunits are present in the mitochondria. Similarly, Kir6.1 (approximately 46 kDa), Kir6.2 (approximately 46 and approximately 40 kDa), and SUR2 (approximately 140 kDa) proteins were found to be expressed in mitochondria using Western blot analysis. By contrast, SUR1 was not present in mitochondria. These results suggest that mitoKATP channels in rat hearts might comprise a combination of Kir6.1, Kir6.2, and SUR2 subunits.

ATP-Binding Cassette Transporters↗

PubMed2005

Tissue microarrays: bridging the gap between research and the clinic.

Tissue microarrays are a high-throughput method for the investigation of biomarkers in multiple tissue specimens at once. This technique allows for the analysis of up to 500 tissue samples in a single experiment using immunohistochemistry and in situ hybridization. Recently, cell lines and xenografts have been reduced to a tissue microarray format and are being applied to preclinical drug development. In clinical research, tissue microarrays are applied at multiple levels: comprehensive analysis of samples in the context of a clinical trial or across a population. Tissue microarrays play a central role in translational research, facilitating the discovery of molecules that have potential roles in the diagnosis, prognosis and prediction of response to therapy.

Animals↗

PubMed2004

Antidepressant effect of Chaihu-Shugan-San extract and its constituents in rat models of depression.

Herbal preparations may be effective alternatives in the treatment of depression, which remains difficult to manage. Chaihu-Shugan-San (CSS), an oriental traditional medicine, has been used as a remedy for Hwa-Byung, a Korean culture-bound syndrome resembling depression. We examined whether aqueous extracts of CSS and its constituent herbs exert antidepressant-like effects in two experimental animal models: the forced swimming test (FST) and the chronic mild stress (CMS) model. The herbal extracts were administered orally for 7 days in the FST and for 21 days during the CMS model; imipramine at 20 mg/kg/day was injected intraperitoneally as a positive control. CSS, Radix Bupleuri (one of the most important constituent plants in CSS), and imipramine had significant anti-immobility effects in the FST and reversed the CMS-induced reduction in sucrose consumption. Rhizoma Cyperi, another constituent of CSS, had antidepressant activity in the FST, while it failed in the CMS model. In conclusion, our results suggest that CSS and its constituent herbs exert antidepressant-like effects comparable to those of imipramine in experimental animal models.

Administration, Oral↗

PubMed2004

Epidermal growth factor-like motifs 1 and 2 of Plasmodium vivax merozoite surface protein 1 are critical domains in erythrocyte invasion.

Plasmodium vivax merozoite surface protein 1 (PvMSP1) is believed to be important in erythrocyte invasion. However, the detailed mechanism of PvMSP1-mediated invasion has been unclear. We demonstrate that the C-terminal 19 kDa domain (PvMSP119) of PvMSP1, the 42-kDa fragment of PvMSP1 is further cleaved to a 33 kDa N-terminal polypeptide and a 19 kDa C-terminal fragment in a secondary processing step, is a critical domain in the binding between parasite ligand and erythrocyte receptor. Also, its cytoadherence was successfully blocked by naturally acquired immunity, was partially sensitive to neuraminidase and trypsin. When expressed separately epidermal growth factor (EGF)-like motifs 1 and 2, subunits of the PvMSP119, mediated 64% and 66% of the erythrocyte-binding activity, respectively, relative to their expression together as a single intact ligand domain. These results suggest that the EGF-like motifs 1 and 2 of PvMSP119 function as a core-binding portion in the attachment of PvMSP1 to erythrocytes.

Animals↗

PubMed2004

Perspectives in tissue microarrays.

Tissue microarrays (TMAs) are means of combining tens to hundreds of specimens of tissue onto a single slide for analysis at one time. TMAs are most frequently constructed from paraffin embedded tissue; however, they can be constructed from frozen tissue. The construction of TMAs is flexible, meeting the focused needs of the investigator. A TMA slide can be processed like an ordinary tissue section, and used for histochemical, immunohistochemical staining or in situ hybridization. Combined with automated new image analysis systems, TMAs are a powerful molecular profiling tool. By confirming the findings of microarray experiments or protein arrays, TMAs can be applied systematically to global cellular network analysis within tissue cell. TMAs are commonly used to confirm the results of expression microarrays as well as in the development of diagnostic and prognostics markers for clinical applications. This review will cover recent advancements in technology for the construction and use of TMAs. Because TMAs can be constructed from archival paraffin embedded tissue, they open up the vast archive of patient samples and make them accessible for medical research. TMAs play an ever increasing role in translational medicine, bridging the chasm of discovery at the research bench to the demonstration of clinical utility prior to implementation in patient care.

Animals↗

PubMed2003

Analysis of the Plasmodium vivax apical membrane antigen-1 gene from re-emerging Korean isolates.

Apical membrane antigen-1 is a candidate vaccine component for malaria. In the present study, we investigated the polymorphism of the Plasmodium vivax apical membrane antigen-1 gene ( PvAMA-1) in 30 Korean isolates. The polymorphic region of the PvAMA-1 gene, corresponding to nucleotides 324-735 (aa 108-245), was amplified using polymerase chain reaction followed by cloning and sequencing. Two genotypes ( SKA and SKG) were identified on the basis of amino acid substitution. These were identical to those of the Chinese isolates. The Korean isolates showed sequence variation at six positions on the basis of the sequence of the Sal1 strain. Of these, variation at position 189 (Glu/Lys) was found only in SKA. These two genotypes were related to the genotype of the circumsporozoite and Duffy binding protein of the Korean isolate. These findings suggest that two genotypes of P. vivax coexist in the endemic area and that the re-emerging parasite in Korea may be related to or have originated in East Asia.

Adult↗

PubMed2003

A multiplex polymerase chain reaction for a differential diagnosis of Plasmodium falciparum and Plasmodium vivax.

A multiplex PCR was designed for the differential diagnosis of the two parasite species by targeting the 18S rRNA gene with a set of primer combinations, amplifying DNA fragments of 1451-bp and 833-bp for P. falciparum and P. vivax, respectively. The sensitivity of this PCR test was high, as minimal as 0.1 parasite per one microliter of blood sample and a minimum of four copies of the target gene could be detected. For the diagnosis of mixed infection of two Plasmodium spp., there were no apparent competition or cross-reaction between the majority and minority Plasmodium species. The multiplex PCR was evaluated on 210 clinical samples and 60 normal controls. The PCR test yielded highly concordant results with microscopic examination, with the only one exception of a mixed (P. falciparum plus P. vivax) infection case, which was diagnosed as a single infection of P. falciparum by microscopy. We propose that the multiplex PCR is a sensitive, specific, and rapid tool that can serve as a useful differential diagnostic tool for detecting P. falciparum and P. vivax.

Animals↗

PubMed2003

Cloning of a pore-forming subunit of ATP-sensitive potassium channel from Clonorchis sinensis.

A complete cDNA sequence encoding a pore-forming subunit (Kir6.2) of ATP-sensitive potassium channel in the adult worm, Clonorchis sinensis, termed CsKir6.2, was isolated from an adult cDNA library. The cDNA contained a single open-reading frame of 333 amino acids, which has a structural motif (a GFG-motif) of the putative pore-forming loop of the Kir6.2. Peculiarly, the CsKir6.2 shows a lack-sequence structure, which deleted 57 amino acids were deleted from its N-terminus. The predicted amino acid sequence revealed a highly conserved sequence as other known other Kir6.2 subunits. The mRNA was weekly expressed in the adult worm.

Adenosine Triphosphate↗

PubMed2002

Taenia solium: identification of specific antibody binding regions of metacestode 10-kDa protein.

Taenia solium neurocysticercosis (NCC) represents one of the major public health problems associated with several neurological manifestations worldwide. We previously identified a recombinant 10-kDa protein of T. solium metacestode (CyDA) specific to active NCC. Immunoblottings with sera from active NCC patients and from animals experimentally infected with larval T. solium (pig), T. saginata (pig), T. asiatica (pig), and T. crassiceps (mouse) strongly recognized CyDA, while sera from patients infected only with adult worms did not. Mapping of antigenic sites using deletion mutants revealed that amino acids (aa) residues 30-34, Asn-Met-Thr-Val-Met (NMTVM), reacted only with sera from active stage T. solium cysticercosis cases. Recognition of CyDA aa 30-34 resided almost exclusively in the IgG4 isotype. Competitive immunoprecipitation with synthetic peptides confirmed the specificity of anti-sera for this penta-peptide. These results demonstrated that aa residues NMTVM in CyDA comprise the core sequence for an active stage NCC-related antigenic determinant. ligand binding protein, HLBP; Cyst fluid, CF; Pooled serum of 10 active NCC patients, serum-pool.

Amino Acid Sequence↗

PubMed2002

Molecular cloning of a pore-forming subunit (Kir6.2 gene) of the ATP-sensitive potassium channel in the bullfrog, Rana catesbeiana Shaw.

A cDNA sequence encoding a pore-forming subunit of ATP-sensitive potassium channel (Kir6.2 gene) of the bullfrog, Rana catesbeiana Shaw, termed RcKir6.2, was isolated from a liver cDNA library. The cDNA contained a single open reading frame of 1,173 bp encoding 391 amino acids with a calculated molecular mass of 42.9 kDa, which has a structural motif (a GFG motif) of the putative pore-forming loop of Kir6.2. Analysis of its phlyogenetic position revealed that the RcKir6.2 is close to Kir6.2 of rabbits. The predicted amino acid sequence shared sequence identity with Kir6.2 of Homo sapiens, Cavia porcellus, Mus musculus, Rattus norvegicus, and Oryctolagus cuniculus by 95.9, 95.6, 96.7, 96.7 and 99.7%, respectively. Expression of RcKir6.2 was detected in various tissues, including heart, kidney, liver, lung, spleen, and stomach of the bullfrog.

ATP-Binding Cassette Transporters↗