Ju-Youn Choi

α-Synuclein Interacts with Phospholipase D Isozymes and Inhibits Pervanadate-induced Phospholipase D Activation in Human Embryonic Kidney-293 Cells

α-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinsons disease and Alzheimers disease. Although the function of α-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of α-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type α-synuclein or the mutant forms of α-synuclein (A53T, A30P) associated with Parkinsons disease. Tyrosine phosphorylation of α-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr125 to Phe slightly increases inhibitory effect of α-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing α-synuclein than in control cells. Binding of α-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Aβ component of α-synuclein. Although biologically important, co-transfection studies indicate that the interaction of α-synuclein with PLD does not influence the tendency of α-synuclein to form pathological inclusions. These results suggest that the association of α-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of α-synuclein.

Alzheimer's disease-associated amyloid beta interacts with the human serine protease HtrA2/Omi.

Amyloid beta (Abeta), a principle component of the cerebral plaques found in the brains of patients with Alzheimers disease (AD), is a pivotal factor implicated in the pathogenesis of AD. Recent reports show that not only extracellular Abeta but also intracellular Abeta induces neuronal apoptosis; however, the mechanism remains to be elucidated. Using yeast two-hybrid assays, we found that Abeta interacts with HtrA2/Omi, an essential human serine protease with proapoptotic activity. Additionally, we mapped the C-terminal region containing the PDZ domain of HtrA2/Omi as the binding determinant for Abeta? The interaction of Abeta with HtrA2/Omi was further confirmed through in vivo co-immunoprecipitation assay in HEK293 cells. This study suggests the possibility that the accumulation of intracellular Abeta and a function of proapoptotic protease, HtrA2/Omi are correlated.

E3 ligase activity of RING finger proteins that interact with Hip‐2, a human ubiquitin‐conjugating enzyme

To identify proteins that interact with Huntingtin‐interacting protein‐2 (Hip‐2), a ubiquitin‐conjugating enzyme, a yeast two‐hybrid screen system was used to isolate five positive clones. Sequence analyses showed that, with one exception, all Hip‐2‐interacting proteins contained the RING finger motifs. The interaction of Hip‐2 with RNF2, one of the clones, was further confirmed through in vitro and in vivo experiments. Mutations in the RING domain of RNF2 prevented the clone from binding to Hip‐2, an indication that the RING domain is the binding determinant. RNF2 showed a ubiquitin ligase (E3) activity in the presence of Hip‐2, suggesting that a subset of RING finger proteins may have roles as E3s.

TGF‐β‐induced cell‐cycle arrest through the p21WAF1/CIP1‐G1 cyclin/Cdks‐p130 pathway in gastric‐carcinoma cells

Transforming growth factor‐β1 (TGF‐β) inhibits cell‐cycle progression of many types of cells by arresting them in G1/S phase through inhibition of the active cyclin‐Cdk complexes that lead to inhibition of Rb phosphorylation. In gastric‐cancer cells, SNU16, TGF‐β treatment induced enhanced expression of p21WAF1/CIP1 (p21), which inhibited the kinase activity of cyclin‐D‐ and cyclin‐E‐associated Cdks and blocked p130 phosphorylation. TGF‐β also enhanced the stability of p130, suggesting that hypophosphorylation of p130 and increased stability of p130 contribute to p130‐mediated G1 arrest in gastric‐cancer cells. Our results demonstrate that p21 and p130 are major downstream targets of TGF‐β in gastric‐cancer cells and that a p21‐G1 cyclin/Cdks‐p130/E2F pathway mediates growth inhibition by TGF‐β in these cells. Int. J. Cancer 83:512–517, 1999.

A 13‐year longitudinal study of the impact of double mutations in the core promoter region of hepatitis B virus on HBeAg seroconversion and disease progression in patients with genotype C chronic active hepatitis

Summary.  The pathogenic role of core promoter (CP) mutations (T1762/A1764) of hepatitis B virus (HBV) in hepatitis B e antigen (HBeAg) seroconversion or disease progression remains unclear. We investigated the clinical relevance of these mutants over a long‐term follow‐up period of up to 15 years. In this longitudinal cohort study, 29 HBeAg‐positive patients with biopsy‐proved chronic active hepatitis without cirrhosis were regularly monitored for >10 years. The viral isolates were characterized, using the frozen liver tissue obtained on the day of biopsy. Long‐term outcomes were compared between patients with and without CP mutations of HBV at baseline. HBV genotyping showed that 100% of study subjects were infected with genotype C HBV. During a median follow‐up period of 12.5 years, patients without double CP mutations of HBV at baseline showed a tendency towards achieving an earlier HBeAg seroconversion than those with (6.9 vs 9.4 years, P = 0.062) double CP mutations. Double CP mutations at baseline were also significantly associated with the eventual development of cirrhosis or hepatocellular carcinoma (P = 0.013), whereas the absence of double CP mutations predicted inactive carrier status at the last follow‐up (P = 0.027). At 10 years, liver‐related tests were also significantly better in patients without double CP mutations of HBV than in those with these mutations, as reflected by higher platelet counts and albumin levels (P = 0.036 and P = 0.044, respectively). Double T1762/A1764 mutations are significantly related to liver deterioration in HBeAg‐positive genotype C active hepatitis patients. A longer period of immune clearance coupled with delayed HBeAg seroconversion appears to contribute to disease progression in patients harbouring these mutations in the CP region of HBV.

Fine epitope mapping of monoclonal antibodies specific to human α-synuclein

Abstract The neuronal phosphoprotein α-synuclein has been increasingly implicated in the pathogenesis of Parkinsons disease (PD) and other neurodegenerative diseases; however, the exact function of α-synuclein still remains illusive. Suitable antibodies (Abs) specific for the gene of interest are indispensable for studying biological and immunological properties of the target gene. Here, we report not only the generation and characterization of monoclonal Abs, Syn-1 and Syn-17, against human α-synuclein, but also the epitope mapping by using recombinant synuclein family proteins and various GST fusion proteins of human α-synuclein domains. Syn-17 recognizes human and rodent α-synuclein, and its epitope is localized within residues 97–99 and 101 of α-synuclein. In contrast, the Syn-1 epitope is localized in residues 121 and 122 of human α-synuclein, and Syn-1 recognizes only human but not rodent α-synuclein, indicating that it can be utilized as a useful reagent for studying human α-synuclein transgenic mouse and zebrafish lines.

Performance of Posttransplant Model for End-Stage Liver Disease (MELD) and Delta-MELD Scores on Short-Term Outcome After Living Donor Liver Transplantation

The performance of the Model for End-stage Liver Disease (MELD) and delta-MELD scores in predicting posttransplant survival has been variable and the results are conflicting, suggesting that posttransplantational factors are more important than pre- or peritransplantational factors in outcomes following liver transplantation (OLT). We assessed the value of posttransplant MELD and delta-MELD scores to predict short-term (90-day) posttransplant survival. We evaluated 279 consecutive patients undergoing living donor OLTs. The MELD scores were calculated serially from pretransplantation as well as 3, 7, and 14 days after transplantation. Twenty-seven (9.7%) among 279 patients died within 90 days after transplantation. Pretransplant MELD score was not associated with short-term posttransplant mortality. The area under the receiver operating characteristic (AUROC) curve in predicting 90-day mortality was 0.637 for posttransplant 3-day MELD, 0.746 for posttransplant 7-day MELD, and 0.859 for posttransplant 14-day MELD score (P = .047, <.001, and <.001, respectively); AUROC was 0.582, 0.646, and 0.784 for 3-day, 7-day, and 14-day delta-MELD scores (P = .235, .034, <.001, respectively). Upon multivariate analysis, posttransplant 14-day MELD (> or =20 vs <20), and 14-day delta-MELD scores (> or =-3 vs <-3) were independent short-term prognostic factors with risk ratios of 18.875 (95% confidential interval [CI]: 4.625-77.028, P < .001) and 13.577 (95% CI: 2.641-69.791, P = .002), respectively. In conclusion, determination of posttransplant 14-day MELD and 14-day delta-MELD scores may afford suitable short-term prognostic predictors for patients following living donor OLT.

Pretreatment HBeAg level and an early decrease in HBeAg level predict virologic response to entecavir treatment for HBeAg-positive chronic hepatitis B.

Summary.  There are few reports on hepatitis B e antigen (HBeAg) titres during nucleos(t)ide analogues treatment. We investigated the changes in HBeAg levels in patients treated with entecavir and the usefulness of HBeAg quantification for predicting antiviral response. Ninety‐five consecutive HBeAg‐positive patients treated with entecavir for more than 48 weeks were enrolled. Serum levels of hepatitis B surface antigen (HBsAg), HBeAg and HBV DNA were assessed at 4‐week intervals to week 24 and thereafter at 12‐week intervals. Virologic response (Y1VR) was defined as an undetectable HBV DNA level at week 48 of therapy. During 48 weeks, HBeAg and HBV DNA level decreased significantly in a biphasic manner and HBsAg level tended to decease. Fifty‐three patients (55.8%) attained Y1VR. Pretreatment HBeAg levels were significantly lower in the Y1VR group than in no Y1VR group. At week 4 and 12 of therapy, 25% and 41.4% of patients showed a decrease of HBeAg levels with >0.5 log10 and >1.0 log10 from baseline, respectively. These patients achieved more Y1VR than those with less decrease of HBeAg levels (97.7%vs 22.2% and 86.2%vs 29.3%, respectively). HBeAg level at week 12 had higher predictive values for Y1VR than HBV DNA level. Multivariate analysis revealed that a pretreatment HBeAg level of <360 PEIU/mL and the reduction in HBeAg level >1.0 log10 at week 12 were associated with Y1VR. These results suggest that pretreatment HBeAg level and an early decrease in HBeAg level are useful measurements for predicting one‐year virologic response during entecavir treatment.

Intraoperative Changes in Hyponatremia as a Risk Factor for Prolonged Mechanical Ventilation After Living Donor Liver Transplantation

Prolonged mechanical ventilation (PMV), a common clinical manifestation, may result in fatal outcomes after living donor liver transplantation (LDLT). Although hyponatremia contributes to neurologic alterations in association with PMV, the effects of acute changes in hyponatremia during LDLT have not been well studied. We sought to determine whether an acute change in hyponatremia during surgery might be a risk factor for PMV after LDLT. Perioperative data were retrospectively collected from 381 patients who underwent LDLT from January 2000 to December 2008. PMV was defined as the need for ≥24 hours of mechanical ventilation within the first postoperative week. Using multivariate logistic regression a simple comparison of perioperative variables between the PMV group and the non-PMV group yielded a predictive model to establish PMV. Thirty-seven patients (9.7%) experienced PMV after LDLT. Intraoperative changes in blood sodium were associated with postoperative PMV; however, the relationship was limited to patients with preoperative hyponatremia. Patients with PMV showed lower survival rates than those without PMV (56.3% vs 86.3%; P <.001). A multivariate analysis revealed that preoperative hepatic encephalopathy, hypotension during surgery (more than 3 bowls), and intraoperative changes in hyponatremia were predictive of PMV. Among the hyponatremia change subgroups, only a severe intraoperative change (≥10 mEq/L) was associated with PMV occurrence (odds ratio, 5.85; 95% confidence interval, 1.62 to 21.20, P = .007). In conclusion, a severe intraoperative change in hyponatremia was a risk factor for PMV in the immediate period after LDLT.

Early development of de novo hepatocellular carcinoma after direct-acting agent therapy: Comparison with pegylated interferon-based therapy in chronic hepatitis C patients

Patients with chronic hepatitis C who achieve a sustained viral response after pegylated interferon therapy have a reduced risk of hepatocellular carcinoma, but the risk after treatment with direct‐acting antivirals is unclear. We compared the rates of early development of hepatocellular carcinoma after direct‐acting antivirals and after pegylated interferon therapy. We retrospectively analysed 785 patients with chronic hepatitis C who had no history of hepatocellular carcinoma (211 treated with pegylated interferon, 574 with direct‐acting antivirals) and were followed up for at least 24 weeks after antiviral treatment. De novo hepatocellular carcinoma developed in 6 of 574 patients receiving direct‐acting antivirals and in 1 of 211 patients receiving pegylated interferon. The cumulative incidence of early hepatocellular carcinoma development did not differ between the treatment groups either for the whole cohort (1.05% vs 0.47%, P = .298) or for those patients with Child‐Pugh Class A cirrhosis (3.73% vs 2.94%, P = .827). Multivariate analysis indicated that alpha‐fetoprotein level >9.5 ng/mL at the time of end‐of‐treatment response was the only independent risk factor for early development of hepatocellular carcinoma in all patients (P < .0001, hazard ratio 176.174, 95% confidence interval 10.768‐2882.473) and in patients treated with direct‐acting agents (P < .0001, hazard ratio 128.402, 95% confidence interval 8.417‐1958.680). In conclusion, the rate of early development of hepatocellular carcinoma did not differ between patients treated with pegylated interferon and those treated with direct‐acting antivirals and was associated with the serum alpha‐fetoprotein level at the time of end‐of‐treatment response.