Biomedical subjects

J H Ou

Publications and source records attributed to J H Ou.

At least 19 recordsLinked to original sources

PubMed2004

A heat inducible tyrosinase with distinct properties from Bacillus thuringiensis.

AIMS: The objective of this work was to purify the tyrosinase from Bacillusthuringiensis subsp. kurstaki (Bt) (CCTCC AB 90010) and study its enzymatic properties. METHODS AND RESULTS: A 'one-step' purification method was used in this work, which was an easy, high-yield purification method. Tyrosinase activity of this purity was measured under different conditions to study its kinetic characterizations. The optimum pH and thermal stability of this enzyme were also determined. The results revealed that the tyrosinase from Bt has distinct properties compared with those from other sources. CONCLUSIONS: A heat-inducible tyrosinase of a wild strain of Bt was identified and partially characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The distinct properties of Bt tyrosinase are important to the application of Bt as a biology pesticide.

Bacillus thuringiensis↗

PubMed2001

Differential regulation of hepatitis B virus gene expression by the Sp1 transcription factor.

The expression of hepatitis B virus (HBV) genes is regulated by a number of transcription factors. One such factor, Sp1, has two binding sites in the core promoter and one in its upstream regulatory element, which is also known as the ENII enhancer. In this study, we have analyzed the effects of these three Sp1 binding sites on the expression of HBV genes. Our results indicate that both Sp1 binding sites in the core promoter are important for the transcription of the core RNA and the precore RNA. Moreover, while the downstream Sp1 site (the Sp1-1 site) in the core promoter did not affect the transcription of the S gene and the X gene, the upstream Sp1 site (the Sp1-2 site) in the core promoter was found to negatively regulate the transcription of the S gene and the X gene, as removal of the latter led to enhancement of transcription of these two genes. The Sp1 binding site in the ENII enhancer (the Sp1-3 site) positively regulates the expression of all of the HBV genes, as its removal by mutation suppressed the expression of all of the HBV genes. However, the suppressive effect of the Sp1-3 site mutation on the expression of the S gene and the X gene was abolished if the two Sp1 sites in the core promoter were also mutated. These results indicate that Sp1 can serve both as a positive regulator and as a negative regulator for the expression of HBV genes. This dual activity may be important for the differential regulation of HBV gene expression.

Binding Sites↗

PubMed2000

Transitional cell carcinoma in dialysis patients.

OBJECTIVE: The aim of our study was to determine whether there is an increased incidence of urothelial cancer, especially transitional cell carcinoma (TCC), in uremic patients on dialysis. METHODS: Retrospective chart analyses were completed for 1,910 uremic patients undergoing maintenance dialysis between January 1987 and December 1997. The incidence of urinary tract cancer was assessed. Only the patients with cancers diagnosed after start of dialysis were enrolled in the study. RESULTS: Of the 1,910 patients, 70 had concomitant urinary tract cancers. Nineteen patients (0.99%), including 17 patients with TCC and 2 patients with renal cell carcinoma, were diagnosed after the initiation of dialysis. The average duration from dialysis to TCC diagnosis was 38.3 (range 2-144) months. Painless gross hematuria was the cardinal symptom in 16 of the 17 patients with TCC. In the 17 patients with TCC, no distant metastases were found at the time of diagnosis. Fourteen patients (82.3%) were stage 0 or A, and 1 patient was stage B1. CONCLUSIONS: The 0.89% incidence of TCC in our dialysis patients was high as compared with that of the general population. The risks of developing urinary TCC in dialysis patients were examined, and we suggest that immunosuppressive stage, dialysis procedure, and chronic bladder irritation (decreased urinary wash effect) may play a part in the development of urinary TCC in dialysis patients. Early detection of hematuria due to regular visits and decreased exposure of urinary tract epithelium to carcinogens from urine may explain why early-stage TCC was seen in most of our patients.

Aged↗

PubMed1999

Activation of p53 tumor suppressor by hepatitis C virus core protein.

In addition to being a structural protein that packages the viral genomic RNA, hepatitis C virus (HCV) core protein possesses regulatory functions. In this report, we demonstrate that the HCV core protein could enhance the gene transactivation activity of the tumor suppressor p53, regardless of whether p53 was derived from an exogenous or an endogenous gene. The activation of p53 by the HCV core protein was supported by the observation that the HCV core protein could enhance the expression of p21(waf1/Cip1), a downstream effector gene of p53, in a p53-dependent manner. Further studies indicated that the HCV core protein could also suppress hepatocellular growth via p53. The HCV core protein and p53 could bind to each other in vitro, which was evidenced by the coimmunoprecipitation, the GST pull-down, and the Far-Western blot assays. The deletion-mapping analysis indicated that the carboxy-terminal sequence of p53 located between amino acids 366 and 380 was required for the core protein binding. These results raised the possibility that the HCV core protein might activate p53 through direct physical interaction. The persistent perturbation of p53 activity by the HCV core protein during chronic infection may have important consequences in HCV pathogenesis.

Carcinoma, Hepatocellular↗

PubMed1999

Mechanism of suppression of hepatitis B virus precore RNA transcription by a frequent double mutation.

A double mutation which converts nucleotide 1765 from A to T and nucleotide 1767 from G to A is frequently found in the hepatitis B virus (HBV) genome isolated from HBV patients with chronic hepatitis symptoms. This double mutation is located in the core promoter that controls the transcription of the precore RNA and the core RNA. In addition, this double mutation also resides in the X protein coding sequence, converting codon 130 from Lys to Met and codon 131 from Val to Ile. Previous studies indicate that this double mutation removes a nuclear receptor binding site in the core promoter, suppresses specifically precore RNA transcription, and enhances viral replication. In this study, we further investigated how this double mutation suppresses precore RNA transcription. We found that this double mutation not only removed the nuclear receptor binding site but also created an HNF1 transcription factor binding site. Further transfection studies using Huh7 hepatoma cells indicate that the removal of the nuclear receptor binding site has no effect on the transcription of HBV RNAs, the two-codon change in the X protein sequence suppresses the transcription of both precore and core RNAs, and the creation of the HNF1 binding site restores the core RNA level. Hence, the specific suppression of precore RNA transcription by this frequent double-nucleotide mutation is the combined result of multiple factors.

Binding Sites↗

PubMed1997

Interaction of transcription factors RFX1 and MIBP1 with the gamma motif of the negative regulatory element of the hepatitis B virus core promoter.

The negative regulatory element (NRE) of the hepatitis B virus (HBV) core promoter contains three subregions which act synergistically to suppress core promoter activity. One of these subregions, NRE gamma, is active in both HeLa cervical carcinoma cells and Huh7 hepatoma cells and was found to be bound by a protein factor present in both cell types. Here we show that the transcription factor RFX1 can bind to NRE gamma and transactivate the core promoter through this site. Mutations which abrogated the gene-suppressive activity of NRE gamma prevented RFX1 from binding to NRE gamma. In addition, RFX1 can bind simultaneously, most likely as a heterodimer, with the transcription factor MIBP1 to NRE gamma. In the absence of a cloned MIBP1 gene for further studies, we hypothesize that RFX1 acts with MIBP1 to negatively regulate the core promoter activity through the NRE gamma site. The ability of RFX1 to transactivate the core promoter raises the possibility that RFX1 may play a dual role in regulating HBV gene expression.

Animals↗

PubMed1997

Effects of a frequent double-nucleotide basal core promoter mutation and its putative single-nucleotide precursor mutations on hepatitis B virus gene expression and replication.

The basal core promoter (BCP) of hepatitis B virus (HBV) directs the transcription of both precore RNA and core RNA which code for e antigen (HBeAg) and core antigen, respectively. A double mutation in the BCP which converts nucleotide (nt) 1762 from A to T and nt 1764 from G to A is frequently observed in patients with chronic hepatitis B. We recently demonstrated that this double mutation prevented the binding of a liver-enriched factor (LEF) to the BCP, suppressed only precore RNA transcription (and hence HBeAg expression), and enhanced progeny virus production. In order to understand the mechanism for the selection of this frequent double mutation, we have extended our previous studies to further characterize LEF and to compare the effects of this double-nucleotide mutation (M1) with each single-nucleotide mutation at nt 1762 (M2) and nt 1764 (M3). Our results indicate that LEF is likely composed of a heterodimer formed between the transcription factor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and an unidentified liver-enriched factor. Further studies reveal that both M1 and M2 prevent the binding of LEF to the BCP, suppress only precore RNA transcription, and increase the efficiency of progeny virus synthesis. In contrast, M3 retains some LEF binding activity, does not suppress HBV RNA transcription, and reduces slightly the efficiency of virus progeny synthesis. The reduced ability of M3 to replicate indicates that it has no selection advantage in itself at the level of the infected hepatocyte. In spite of its enhanced replication rate, M2 is rarely detected in HBV patients. This indicates the involvement of factors other than intracellular replication rates in the selection of these virus variants in the infected individual.

Adenine↗

PubMed1997

Molecular biology of hepatitis B virus e antigen.

Hepatitis B virus (HBV) e antigen (HBeAg) was discovered in 1972 as one of the serological markers of HBV infection. Although 25 years have passed since its initial discovery, the function of this antigen in the life cycle of HBV has remained elusive. Mutations in the HBV genome that prevent the expression of HBeAg do not abolish the replication of HBV, indicating that this antigen is not essential for HBV replication. In contrast, the conservation of the HBeAg gene in the genomes of related animal viruses, including the distantly related duck HBV, argues for an important function of this antigen. The purpose of the present article is to review the molecular biology of HBeAg and to examine its possible functions in the life cycle of HBV.

Animals↗

PubMed1997

Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor.

Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV.

Binding Sites↗

PubMed1996

Characterization of nuclear localization of a hepatitis B virus precore protein derivative P22.

Both of hepatitis B virus core protein and a precore protein derivative, named P22, have been shown to localize in the nucleus. Although P22 has ten additional amino acid residues at its amino-terminus, both proteins contain the same nuclear localization signal. In order to understand the mechanism that regulates the activity of this signal, we have studied the nuclear localization of P22 and compared it with that of core protein. It was found that both cytosolic and nuclear fractions of P22 were phosphorylated but to a lesser extent when compared with cytosolic core protein. This distinction was likely attributed to different conformations between these two proteins since the density gradient analysis revealed a different particle formation for P22 in the cytosol. When expressed in Vero cells synchronized by serum deprivation, P22 remained in the cytosol during G0 and G1 phases, accumulated gradually in the nucleus during S phase, and largely localized in the nucleus when cells were confluent. On the other hand, the core protein was transported into the nucleus during mid-G1 phase, shuttled back to the cytosol in S phase and again accumulated in the nucleus when cells were confluent. Interestingly, when aphidicolin was used to arrest the cells in late G1 phase, both proteins were found to accumulate in the nuclei. These results indicated that although both P22 and core proteins possessed the same nuclear localization signal, the cellular regulation of their nuclear transport was not identical and might involve different molecular mechanisms.

Animals↗

PubMed1996

Interaction between hepatitis C virus core protein and E1 envelope protein.

Hepatitis C virus has three structural genes named C, E1, and E2. The C gene encodes the core (capsid) protein and the E1 and E2 genes encode the envelope proteins. In an immunoprecipitation experiment, the E1 protein was found to be precipitated by an anti-core antibody in the presence but not in the absence of the core protein, indicating that the E1 protein can interact with the core protein. This interaction is independent of whether the E1 and the C genes are linked in cis or separated in different DNA constructs for expression. The interaction between the core and the E1 proteins is confirmed by the observation that a hybrid protein derived from the core protein and the tissue plasminogen activator is localized in the nucleus in the absence of the E1 protein and in the perinuclear region in the presence of the E1 protein. Deletion-mapping studies indicate that the carboxy-terminal sequences of both the core and the E1 proteins are important for their interaction. Since little E1 sequence is exposed on the cytosolic side of the membrane of the endoplasmic reticulum, the interaction between the core and the E1 proteins most likely takes place in the endoplasmic reticulum membrane. The E2 protein could not be coprecipitated with the core protein by the anti-core antibody in a similar assay and likely does not interact with the core protein. The implications of these findings on the morphogenesis of the hepatitis C virus virion are discussed.

Chromosome Mapping↗

PubMed1996

Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication.

The basal core promoter (BCP) of hepatitis B virus (HBV) controls the transcription of both the precore RNA and the core RNA. The precore RNA codes for the secreted e antigen, while the core RNA codes for the major core protein and the DNA polymerase and also is the pregenomic RNA. The double mutation of nucleotides 1762 and 1764 in the BCP from A and G to T and A, respectively, is frequently observed in HBV sequences isolated from chronic patients. Several papers have reported conflicting results regarding whether this double mutation is important for e antigen expression. In order to address this issue, we have introduced this double mutation into the HBV genome and studied its effects on HBV gene expression and replication. Our results indicate that the mutated BCP can no longer bind a liver-enriched transcription factor(s) and that the transcription of only precore RNA and, consequently, the expression of e antigen were reduced. The reduction of precore gene expression was accompanied by an increase in progeny virus production. This increase was found to occur at or immediately prior to the encapsidation of the pregenomic RNA. Thus, the results of our in vitro study resolve the discrepancy of previous clinical observations and indicate that this double mutation suppresses but does not abolish the e antigen phenotype. The implications of these findings in the pathogenesis of HBV are discussed.

Animals↗

PubMed1995

Cell type-dependent regulation of the activity of the negative regulatory element of the hepatitis B virus core promoter.

The Hepatitis B virus core promoter regulates the expression of the core protein, the precore protein, and the viral DNA polymerase. This promoter is transactivated by HNF4, a liver-enriched transcription factor, through an HNF4 binding site located upstream of the core promoter. The transactivation activity of HNF4 on the core promoter is antagonized by a negative regulatory element (NRE) located upstream of the HNF4 binding site. While the NRE can effectively antagonize HNF4 to suppress the core promoter in HeLa cervical carcinoma cells, it has only a marginal suppressing activity on the core promoter in Huh7 hepatoma cells. By performing deletion-mapping experiments, we have found that the NRE contains at least three independent subregions named NRE alpha, NRE beta, and NRE gamma. Each of these three subregions possesses a weak suppressing activity, but together they generate a strong synergistic suppressing effect on the core promoter. The NRE gamma subregion is active in both HeLa and Huh7 cells and is bound by a protein factor slightly less than 130 kDa in molecular mass. The NRE alpha and NRE beta subregions are active in HeLa cells but not in Huh7 cells. Thus, the marginal suppressing effect of the NRE observed in Huh7 cells was mostly due to the activity of the NRE gamma subregion. No clear protein factor binding sites could be identified in the NRE alpha and NRE beta subregions when the HeLa nuclear extract was used for the DNaseI-footprinting analysis, indicating weak or no protein association with these two subregions in this cell type. However, extensive protein factor binding sites could be identified throughout the sequences of these two subregions when the Huh7 nuclear extract was used for the analysis. These results indicate that a different set of protein factors binds to the NRE alpha and NRE beta subregions in Huh7 cells and may account for the inactivity of these two subregions in this cell type. Thus, our results indicate that the cell type-dependent activity of the NRE is due to differential regulation of the activities of the NRE alpha and NRE beta subregions by the cell types. This regulation is most likely mediated by cell type-dependent protein factors.

Base Sequence↗

PubMed1995

Differential subcellular localization of hepatitis C virus core gene products.

The expression of the core gene of two different hepatitis C virus (HCV) isolates was analyzed. In the presence of its downstream E1 envelope protein sequence, two major core protein products with molecular masses of 21 kDa (P21) and 19 kDa (P19) and a minor protein product with molecular mass of 16 kDa (P16) were detected. In the absence of its downstream E1 envelope protein sequence, P21 and P19 remained the major protein products expressed from the core gene of the HCV-RH isolate, whereas P16 became the major protein product of the core gene of the HCV-1 isolate. Analysis of the amino-terminal sequences of P21 and P16 expressed in Escherichia coli revealed that P21 and P16 were co-amino terminal. Deletion-mapping analysis indicated that P16 lacked the carboxy-terminal sequence of P21. Immunofluorescence analysis of the subcellular localization of different HCV core proteins indicated that P21 and P19 displayed a reticular and punctate staining pattern typical of endoplasmic reticulum-associated proteins, while P16 was localized to the nucleus. The distinct subcellular localization of P16 raises the possibility that P16 may have a biological function very different from those of P21 and P19.

Amino Acid Sequence↗

PubMed1995

Transoral anterior decompression and fusion of chronic irreducible atlantoaxial dislocation with spinal cord compression.

STUDY DESIGN: In this study, 10 patients with chronic irreducible atlantoaxial dislocation were treated by transoral anterior decompression and fusion. OBJECTIVES: To examine the benefits of the transoral approach, the patients treated with this procedure were compared with the historical control subjects after 2 years of follow-up. SUMMARY AND BACKGROUND DATA: Chronic irreducible atlantoaxial dislocation with cord compression is difficult to treat because the cord is compressed posteriorly by the posterior arch of the atlas as well as anteriorly by the posterior-superior portion of the axial body and nonunited dens. Its irreducibility, as a result of the bony scarring between the dens and the anterior body of the axis, and the locking of the lateral joints of C1-C2, makes reduction more complex. Posterior surgical approaches have been associated with high morbidity and mortality. METHODS: Ten patients were diagnosed and followed up by clinical symptoms, radiography, pantopaque myelography, and computed tomography. They were treated surgically by transoral decompression and fusion. During the surgery the nonunited dens as well as callus, granulation, and scar tissue were removed; the cartilage of the articular surfaces of the atlantoaxial joint was excised. Postoperative treatment included skull-cervical biaxial traction, tracheostomy care, nasal feeding, and Minerva cast. RESULTS: The 2- to 6-year follow-up showed that four out of 10 patients recovered completely and returned to work, three recovered to a great degree and ambulated, two partially recovered, and one recovered poorly. CONCLUSION: Transoral decompression and fusion offered satisfactory results in a series of patients with chronic irreducible atlantoaxial dislocation. None of the patients showed serious complications of stability, even though only one had a secondary posterior fusion. Therefore, anterior decompression associated with subtotal obliteration of the atlantoaxial joints without bone grafts is a feasible therapy for irreducible atlantoaxial dislocation using a multifunctional bed and biaxial traction.

Adolescent↗

PubMed1995

Key role of a CCAAT element in regulating hepatitis B virus surface protein expression.

Two separate promoters, the upstream preS1 and the downstream S promoters, give rise to transcripts encoding three forms of the hepatitis B virus surface protein. Overproduction of large surface protein because of increased preS1 transcripts leads to a block in secretion of all forms of the surface protein and of virion particles. We show here that a CCAAT element in the S promoter not only increases the amount of S transcripts, but also decreases the amount of preS1 transcripts by up to fivefold. Consequently, mutations in this element cause intracellular accumulation of surface proteins because of the secretory block. Therefore, this CCAAT element appears to be critical for maintaining the high ratio of S versus preS1 transcripts that is necessary for the viral life cycle.

Base Sequence↗

PubMed1995

Phosphorylation and nuclear localization of the hepatitis B virus core protein: significance of serine in the three repeated SPRRR motifs.

Hepatitis B virus core protein (antigen) is an important serologic marker of hepatitis B virus infection. This protein is found in the cytoplasm or the nuclei, or both, of infected hepatocytes. A nuclear localization signal has previously been identified in the core protein sequence. This signal overlaps three repeated SPRRR motifs. In this report, we demonstrate that substitution of all of the serine residues in these three SPRRR motifs with alanine can prevent almost entirely the phosphorylation of the core protein in Huh-7 hepatoma cells, enhance nuclear localization of the core protein in both Huh-7 and nonhepatic cells, and abolish cell cycle regulation of nuclear localization of the core protein. Since the three core protein mutants which retained only one serine residue of each of the three SPRRR motifs could be phosphorylated to similar degrees, these three serine residues likely could serve as the acceptor sites for phosphorylation with equal efficiency. These results, together with the observation that the three SPRRR motifs overlap the nuclear localization signal of the core protein, raise the possibility that nuclear localization of the core protein is negatively regulated by phosphorylation of the serine residues in the SPRRR motifs.

3T3 Cells↗

PubMed1994

Regulation of hepatitis B virus ENI enhancer activity by hepatocyte-enriched transcription factor HNF3.

Hepatitis B virus (HBV) ENI enhancer can activate the expression of HBV and non-HBV genes in a liver-specific manner. By performing the electrophoretic mobility-shift assays, we demonstrated that the three related, liver-enriched, transcription factors, HNF3 alpha, HNF3 beta, and HNF3 gamma could all bind to the 2c site of HBV ENI enhancer. Mutations introduced in the 2c site to abolish the binding by HNF3 reduced the enhancer activity approximately 15-fold. Moreover, expression of HNF3 antisense sequences to suppress the expression of HNF3 in Huh-7 hepatoma cells led to reduction of the ENI enhancer activity. These results indicate that HNF3 positively regulates the ENI enhancer activity and this regulation is most likely mediated through the 2c site. The requirement of HNF3 for the ENI enhancer activity could explain the liver specificity of this enhancer element.

Base Sequence↗