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Biomedical subjects

Y R Hong

Publications and source records attributed to Y R Hong.

At least 19 recordsLinked to original sources
PubMed2004

Rac1 gene mutations in human brain tumours.

AIMS: Rac1 is a member of the Ras superfamily of small GTPase and plays a fundamental role in cytoskeleton reorganization, regulation of gene expression and cell proliferation, and cellular transformation. Though recent studies point to an involvement of rac1 in tumorigenesis, little is known about the alteration of rac1 gene in human brain tumours. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), TA cloning, and DNA sequencing were performed to detect rac1 gene mutations in the surgical specimens of 45 human brain tumours. RESULTS: Twelve of 45 cases had base changes in the rac1 gene. The frequency of rac1 alterations was seven of 18 meningiomas, three of 14 astrocytomas, one of seven pituitary adenomas, and one of four metastatic brain tumours. No mutation was detected in acoustic neurilemomas. The subtypes of seven meningiomas include three meningotheliomatous, two atypical, one transitional and one angioblastic meningioma. Three astrocytomas had rac1 gene mutation, including one grade II, one grade III, and one grade IV astrocytoma. All of single base changes were transitions, five of them being T to C transitions. Sites of rac1 mutation were found in codons 34, 41 (two cases), 42 (two cases), 43, 44, 46 and 58. These mutations are mainly localized in the putative effector-domain of rac1 gene and may enhance the activity of rac1, which increases the survival of brain tumours. CONCLUSION: Our results suggest that rac1 gene may play a role in some brain tumours of divergent histogenesis and that the alterations of rac1 gene may contribute to tumorigenesis and/or metastasis.

Adolescent↗
PubMed2001

Boundary sequences of the NADPH oxidase p67(phox) C-terminal SH3 domain play on its specificity.

SH3 domains are found in many signal transduction proteins where they mediate protein-protein binding by recognizing specific peptides rich in proline. Based on the analysis of sequence alignment data, the NADPH oxidase p67(phox) C-terminal SH3 domain possesses a typical compact beta-barrel consisting of five beta-strands arranged in two antiparallel beta-sheets of three and two beta-strands. Multiple amino acid substitutions were made at beta e and its flanking residues to determine the role of the boundary sequences in binding activity and conformational specificity of the domain. Analysis of amino acid P55 indicated that all mutants were completely abolished in their binding activities. The substitution of F58 with Y58 showed no effect of the binding, whereas substitution with stop codon abolished activity. Furthermore, when amino acid V59 was substituted with stop codon, activity was also completely abolished. Substitution of E60 with stop codon showed no effect of binding. Moreover, our data show that V59 particularly could not be replaced by Leu. Taken together, these data suggest that V59 may not only contribute the exact boundary site but also play on the specificity for protein-protein interactions in phagocyte NADPH oxidase.

Amino Acid Sequence↗
PubMed2001

Identification of two down-regulated genes in rat liver allografts by mRNA differential display.

Total RNA differential display (DD) using random primers was performed for rat orthotopic liver transplantation (OLT) models. DA (RT1a) donor livers were transplanted into DA, PVG (RT1c), and LEW (RT1l) recipients: (1) syngeneic OLT (DA-DA): no rejection occurs; (2) allogeneic OLT (DA-PVG): rejection occurs, but is naturally overcome without immunosuppression; (3) allogeneic OLT (DA-LEW): animals die of acute rejection within 14 days. cDNA was isolated from selected bands, re-amplified for sequencing, and confirmed by Northern blots. Two down-regulated genes were observed in day-7 allogeneic OLT livers (DA-PVG, DA-LEW), while they were consistently expressed in day-7 syngeneic OLT (DA-DA) livers. These two genes were identified as alpha-glutathione sulfotransferase (alpha-GST) Ya gene and estrogen sulfotransferase (EST), respectively. Northern blots confirmed that their expression was down-regulated in OLT (DA-PVG) livers on days 7-26 and gradually restored. The mRNA expression of GST and EST may be good markers to predict rejection or induction of tolerance.

Animals↗
PubMed2000

Genomic organization and molecular characterization of the human ninein gene.

The centrosome plays a key role in the formation of the mitotic spindle, cell polarity, and cell locomotion. Previously we identified a novel centrosomal associated protein hNinein using GSK-3beta as a bait in the yeast two-hybrid assay. In this report, the hNinein genome was found to correspond to 29 exons of genomic sequence on human chromosome 14q22. Promoter analysis predicts that hNinein contains a TATA, two CCAAT, and three GC boxes. The promoter exhibits the following potential transcription factor binding sites: Sp1, p300, and AP-1. In addition, an alternatively spliced isoform, encoded a 2041-amino-acid protein of 237,900 Da, which was designated hNinein-Lm (GenBank AF302773). The hNinein-Lm genome was found to correspond to 28 exons (2'-29). Amino acid sequence comparison with hNinein showed that hNinein-Lm exhibited an EF-hand Ca2+ binding domain in the N-terminus which similar to mouse ninein. Northern blot showed that this hNinein-Lm isoform was expressed more than hNinein in tissues examined. Differential RT-PCR combining Southern blotting also showed that hNinein-Lm is much more abundant compared to hNinein. Two forms of ninein may also imply the status of ninein associated with a pair of the centrioles in the centrosome structure. Furthermore, molecular characterization shows that human ninein is oligomerized at the C-terminal end which overlapped with GSK-3beta binding site, suggesting that oligomerization of ninein may be regulated by GSK-3beta phosphorylation.

Alternative Splicing↗
PubMed2000

Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3beta.

Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein. When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa. The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site. Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome. In addition, hNinein also showed to react with centrosomal autoantibody sera. Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.

Amino Acid Sequence↗
PubMed2000

Three rat brain alternative splicing dynamin-like protein variants: interaction with the glycogen synthase kinase 3beta and action as a substrate.

Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have shown that it may be involved in the formation of coated vesicles. In this report, three different alternatively spliced dynamin-like protein variants (DLP1-WT, -11, and -37) from rat brain were identified by reverse transcription/polymerase chain reaction (RT-PCR). One novel rat alternatively spliced variant (DLP1-37), not described previously, was identified. We examined the interaction of these three rat brain dynamin-like protein variants with glycogen synthase kinase 3beta (Gsk-3beta) using the yeast two-hybrid screening, in vitro binding assay, and immunoprecipitation analysis. It was found that all three examined rat brain dynamin-like protein variants can bind to Gsk-3beta. Moreover, in vitro kinase (phosphorylation) assay showed that mammalian dynamin-like protein acts as a substrate for glycogen synthase kinase 3beta. These data suggest that Gsk-3beta may participate in a functional role in dynamin-like proteins in vesicle trafficking.

Alternative Splicing↗
PubMed2000

Differential expression of four human dynamin-like protein variants in brain tumors.

Dynamin-like protein, a large GTP-binding protein, has recently been cloned, and studies have suggested that it is involved in the formation of coated vesicles. In this report, the differential expression of four human dynamin-like protein splice variants (HdynIV-wildtype [WT], -11, -26, and -37) from various brain tumors was identified by reverse transcription/polymerase chain reaction (RT-PCR). One novel variant (HdynIV-11), not described previously, was identified. The four alternatively spliced variants exhibited tissue specificity in normal tissues. The HdynIV-WT was strongly expressed in the brain, whereas HdynIV-37 was expressed in all tissues examined. Moreover, HdynIV-26 was dominant in the liver and apparently overexpressed in all astrocytomas and most meningiomas and adenomas. This report suggests that HdynIV-26 may cause aberrant protein trafficking and alter vesicle formation in brain tumors. Our results also suggest that dynamin-like protein is associated with various brain tumors and, more importantly, that aberrant expression of the HdynIV-26 variant may play a role in brain tumorigenesis.

Adenoma↗
PubMed2000

Clinical significance of distribution patterns of P53 immunoreactivity in astrocytic tumors.

The difference of prognosis in patients with the same WHO grade of astrocytic tumors suggests that such tumors comprise a heterogeneous group in biological behavior. The correlation between p53 immunoreactivity and prognosis has often been reported but remains controversial. From the perspective of clonal expansion of p53 immunopositive cells, the distribution patterns of p53 immunoreactivity can be divided into four types: negative, scattered, focally clustered, and diffusely clustered. The survival rate was evaluated by classifying the p53 immunoreactivity into two groups: the significantly immunopositive patterns (focally and diffusely clustered types) and the significantly immunonegative patterns (negative and scattered types). The survival analysis showed a highly significant difference between these two patterns within the same WHO grade of astrocytic tumors (p = 0.0185). Our studies demonstrate that the distribution patterns of p53 immunoreactivity, which reflect the trends of clonal expansion of p53 immunopositive cells, are significantly valuable in predicting the prognosis of patients with the same WHO grade of astrocytic tumors.

Adult↗
PubMed1999

Expression and mutagenesis studies of cobrotoxin from Taiwan cobra.

The cDNA encoding cobrotoxin was constructed from the cellular RNA isolated from the venom glands of Naja naja atra (Taiwan cobra). The cDNA was subcloned into the expression vector pET20b(+) and transformed into BL21(DE3) Escherichia coli strain. Expressed cobrotoxin was isolated from inclusion bodies of E. coli and subjected to refolding into its folded structure. The refolded cobrotoxin was purified by high-performance liquid chromatography and exhibited a neurotoxicity in inhibiting acetylcholine-induced muscle contractions. Recombinant cobrotoxin showed a tendency to isomerize its disulfide bonds as that observed with native cobrotoxin. An appreciable decrease in the rate of isomerization reaction was observed when Glu-38 was replaced with Gln-38 or Lys-47 was replaced with Glu-47 or Gln-47. These results reflect that the element in controlling the disulfide isomerization of cobrotoxin is closely associated with the charged side chains in the cobrotoxin molecule.

Acetylcholine↗
PubMed1999

Expression and mutation analysis of the p53 gene in astrocytoma.

The role of p53 gene mutations in the formation or progression of human astrocytic tumors is controversial. We studied the distribution pattern of p53 immunoreactivity and analyzed p53 gene mutations to define the significance of p53 gene mutations in astrocytoma tumorigenesis or malignant progression. Twenty-three astrocytic tumors were evaluated with immunohistochemistry, single-strand conformation polymorphism (SSCP) analysis, and sequence analysis. We also searched MEDLINE to collect data on p53 gene mutation frequencies in astrocytic tumors in order to evaluate the association of p53 mutations and tumor grade. Strong immunoreactivity with a diffuse clustering pattern was found in three of five glioblastomas and seven of 12 anaplastic astrocytomas. Three of four low-grade astrocytomas were immunonegative. The p53 immunopositive cells in the only positively staining low-grade astrocytoma in our study appeared sparsely scattered. The results of immunostaining suggested that clonal expansion was associated with astrocytoma progression. Mutations of the p53 gene were detected in four of the 23 astrocytomas (one glioblastoma and three anaplastic astrocytomas). In the genetic data analysis, 76 of 367 astrocytomas had p53 gene mutations. A significantly greater p53 gene mutation frequency was found in anaplastic astrocytomas or glioblastomas than in the low-grade astrocytomas. The results of these immunohistochemical and genetic studies support the view that p53 gene mutation is associated with the malignant progression from low-grade to high-grade astrocytomas rather than with tumor initiation or promotion.

Adolescent↗
PubMed1998

Human dynamin-like protein interacts with the glycogen synthase kinase 3beta.

Members of the dynamin superfamily are implicated in vesicle trafficking. Using human glycogen synthase kinase 3 beta (Gsk-3 beta) as bait in the yeast two-hybrid system, we identified a novel human dynamin-like protein IV (HdynIV). When the full-length cDNA of HdynIV was sequenced, it showed that HdynIV's carboxyl terminal lacks a proline-rich domain that can bind to Gsk-3 beta. By Northern blot analysis and isoform-specific PCR, we found that HdynIV is expressed ubiquitously in all human tissues examined. Two transcripts of 2.4 and 4.4 kb are shown to be more abundant in heart, brain, and skeletal muscle. Interestingly, the 2.4-kb transcript is expressed more distinctly in the fetal liver than in the adult liver, suggesting that this protein might play a role during development. In the present report, we have demonstrated that HdynIV interacts with the Gsk-3 beta through its carboxyl-terminal region, implying than HdynIV may also be involved in cell signaling.

Adult↗
PubMed1998

Phagocyte NADPH oxidase p67-phox possesses a novel carboxylterminal binding site for the GTPases Rac2 and Cdc42.

Rac GTPases regulate activation of the phagocyte NADPH oxidase, a multi-component enzyme complex that produces superoxide in response to host infection. GTP-bound Rac binds to the cytosol protein p67-phox enabling it to participate in oxidase assembly. Details of this interaction are poorly understood. Previous studies showed that Rac/p67-phox binding is GTP-dependent and that several Rac1 mutants lost the ability to activate the oxidase even though they still bound p67-phox. Using two hybrid and blot overlay binding methods, we identified a novel binding site in the p67-phox C-terminus that binds Rac1, Rac2, and Cdc42, a related GTPase which does not activate the oxidase. Binding was independent of the GDP/GTP state. We also showed that GTP-Cdc42 binds p67-phox N-terminus similar to GTP-Rac. Therefore, Rac binding to p67-phox is not synonymous with NADPH oxidase activation, and Rac probably participates in other steps of oxidase activation in addition to binding p67-phox.

Binding Sites↗
PubMed1998

Prognostic evaluation in supratentorial astrocytic tumors using p53, epidermal growth factor receptor, c-erbB-2 immunostaining.

Molecular pathology may play an important role in predicting the tumor prognosis, particularly p53, epidermal growth factor receptor (EGFR), and c-erbB-2. We investigated six variables (age, sex, histopathological grade, p53, EGFR, and c-erbB-2) to identify the role of such factors in predicting the outcome of patients with supratentorial astrocytic tumors. Thirty-seven tumors were studied including 9 well-differentiated astrocytomas (WHO grade 2), 19 anaplastic astrocytomas (WHO grade 3), and 9 glioblastomas multiforme (WHO grade 4). In univariate analysis, no statistical significance was found for the prognostic value of the sex (p = 0.2188), age (p = 0.5530), p53 immunostain (p = 0.2194), and c-erbB-2 immunostain (p = 0.4203). A significant correlation with the prognosis was found with respect to the histopathological grade (p = 0.0049) and EGFR expression (p = 0.0284). In multivariate analysis, the histopathological grade was shown to be significant independent variable (p = 0.0152). In WHO grade 2 and 3 astrocytomas, expression of p53 or EGFR was associated with poorer patient outcome. In glioblastomas, expression of p53 was also associated with poorer prognosis. Our studies suggested that conventional histological assessment of supratentorial astrocytic tumors remains the best guide to prognosis. Although no statistical significance was found between the immunostains and survival in variant grades of astrocytomas, there was a trend between p53 or EGFR proteins expression and the decrease of survival time.

Adult↗
PubMed1996

Elimination of false negative results in the two-hybrid system in the phagocyte NADPH oxidase.

The yeast two-hybrid system is finding increased use in the study of interactions between proteins. In this method, two polypeptides are expressed in yeast as fusion proteins to a transcriptional activator DNA-binding domain (bd) and activating domain (ad), respectively. Interaction between the two polypeptides reconstitutes function of a transactivator which controls expression of reporters. The phagocyte NADPH oxidase is a complex of membrane cytochrome b558 (comprised of subunits p22-phox and gp91-phox) and three cytosol proteins (p47-phox, p67-phox, and p21rac) that translocate to membrane and bind to cytochrome b558. This is the first report to demonstrate that two of cytosolic components of cytochrome b558, p47-phox binding to p67-phox each other. We encountered several methodological problems in the two-hybrid system which are the focus of this report.

False Negative Reactions↗
PubMed1996

Bacteriophage T4 expression-packaging-processing vector that encapsidates HIV-type I GP120-V3 fusion protein inside the head.

Bacteriophage T4 as an expression-packaging-processing vector has recently been developed by using the T4 non-essential capsid scaffold protein IPIII(1-3). The IPIII gene was expressed at high level in E. coli from plasmid and was truncated at its C-terminus to permit construction of gene fusion in three different reading frames of EP-16 vector. Regulated higher-expression PPL reading frame vectors were also constructed. Infection of the plasmid-containing bacteria with bacteriophage mutants deleted for the IPIII gene showed that viable phage encapsidated and proteolytically processed the IPIII fusion proteins. An IPIII gene fused to human immunodeficiency virus type I (HIV-1) gp120 loop3 domain (V3) IPIII-V3 fusion gene products has been constructed and was packaged and processed within viable phage particle. The packaged fusion protein Gp120-V3 may be used for production of vaccine in the future.

Bacteriophage T4↗
PubMed1995

Protection from proteolysis using a T4::T7-RNAP phage expression-packaging-processing system.

DNA coding for bacteriophage T7 RNA polymerase (T7-RNAP) was inserted into a positive selection-vector form of the T4 genome, placing it under the control of bacteriophage T4 ipIII promoters. The recombinant T4::T7-RNAP fusion phage retained infectivity and produced T7-RNAP in infected cells. Fusion genes were constructed by insertion into a plasmid containing an iPIII (encoding internal protein III) target portion and a bacteriophage T7 promoter region. When Escherichia coli cells containing the plasmid were infected with the T4::T7-RNAP re-phage, the bacteria produced fusion protein at high levels. The newly synthesized T4::T7-RNAP re-phage progeny package and process the fusion protein into the phage capsid during head morphogenesis. In this paper, we demonstrate that truncated T4 internal protein IPIII, human IPIII::beta Glo (beta-globin) fusion protein, E. coli IPIII::beta Glo::beta Gal (beta-galactosidase) triple-fusion protein and IPIII::V3 fusion protein (human immunodeficiency virus envelope protein gp120 V3 region) are expressed at high levels by T4::T7-RNAP induction. With IPIII::beta Glo, expression-packaging-processing (EPP) occurs simultaneously with T4::T7-RNAP re-phage infection. We also demonstrate that T4::T7-RNAP re-phage stabilize unstable proteins such as the X90 fragment of beta Gal, thought to be degraded by the lon protease. An unstable 20-kDa fragment of the large subunit of human cytochrome b558, an integral membrane protein in phagocytes, is subject to proteolytic degradation even when produced in the lon-deficient BL21 strain. However, upon induction with T4::T7-RNAP re-phage, the 20-kDa protein is produced intact.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage T4↗