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1.  The herpes simplex virus type 1 major capsid protein (VP5-UL19) promoter contains two cis-acting elements influencing late expression. 
Journal of Virology  1994;68(9):5738-5747.
The herpes simplex virus type 1 (HSV-1) major capsid protein VP5 gene (UL19) is expressed with beta gamma (gamma 1 [leaky late]) kinetics. We have previously described the construction of recombinant HSV-1 in which the VP5 promoter was engineered to control the expression of the bacterial beta-galactosidase gene as a reporter (C.-J. Huang, S. A. Goodart, M. K. Rice, J. F. Guzowski, and E. K. Wagner, J. Virol. 67:5109-5116, 1993). Here we describe further mutational analysis in recombinant viruses. We have precisely defined the boundaries of the VP5 promoter and identified two regions important for both the level and the kinetics of expression. The 5' boundary was located at -48 relative to the initiation site of transcription by analyzing a series of nested deletions in the upstream sequence, and although a number of cis-acting sites influencing transient expression have been identified upstream of this point, these sites have no role in promoter activity during productive infection. Deletion of an Sp1-binding site located between -48 and the TATA box at -30 greatly reduced VP5 promoter activity late but not early after infection. A cis-acting element whose sequence resembles the human immunodeficiency virus type 1 initiator was located between -2 and +10 in the VP5 sequence by characterizing a series of deletions and site-directed block mutations downstream the TATA box. This element defines the 3' limit of the VP5 promoter, and like the upstream element, disruption of this element also inhibited promoter activity late in the productive cycle.
PMCID: PMC236977  PMID: 8057455
2.  Sequence analysis and expression of the Salmonella typhimurium asr operon encoding production of hydrogen sulfide from sulfite. 
Journal of Bacteriology  1991;173(4):1544-1553.
A chromosomal locus of Salmonella typhimurium which complements S. typhimurium asr (anaerobic sulfite reduction) mutants and confers on Escherichia coli the ability to produce hydrogen sulfide from sulfite was recently cloned (C. J. Huang and E. L. Barrett, J. Bacteriol. 172:4100-4102, 1990). The DNA sequence and the transcription start site have been determined. Analysis of the sequence and gene products revealed a functional operon containing three genes which have been designated asrA, asrB, and asrC, encoding peptides of 40, 31, and 37 kDa, respectively. The predicted amino acid sequences of both asrA and asrC contained arrangements of cysteines characteristic of [4Fe-4S] ferredoxins. The sequence of asrB contained a typical nucleotide-binding region. The sequence of asrC contained, in addition to the ferredoxinlike cysteine clusters, two other cysteine clusters closely resembling the proposed siroheme-binding site in biosynthetic sulfite reductase. Expression of lacZ fused to the asr promoter was repressed by oxygen and induced by sulfite. Analysis of promoter deletions revealed a region specific for sulfite regulation and a second region required for anaerobic expression. Computer-assisted DNA sequence analysis revealed a site just upstream of the first open reading frame which had significant homology to the FNR protein-binding site of E. coli NADH-linked nitrite reductase. However, asr expression by the fusion plasmid was not affected by site-specific mutations within the apparent FNR-binding site.
PMCID: PMC207294  PMID: 1704886
3.  General anaesthesia is associated with increased risk of surgical site infection after Caesarean delivery compared with neuraxial anaesthesia: a population-based study 
BJA: British Journal of Anaesthesia  2011;107(5):757-761.
This study compared the odds ratio (OR) of surgical site infection (SSI) within 30 days after operation with general anaesthesia (GA) or neuraxial anaesthesia (NA) in Taiwanese women undergoing Caesarean delivery (CD).
An epidemiologic design was used. The study population was based on the records of all deliveries in hospitals or obstetric clinics between January 2002 and December 2006 in Taiwan. Anonymized claim data from the Taiwan National Health Insurance Research Database (NHIRD) were analysed. Women who received CD were identified from the NHIRD by Diagnosis-Related Group codes. The mode of anaesthesia was defined by order codes. Multivariate logistic regression was used to estimate the OR and associated 95% confidence interval (CI) of post-CD SSIs for GA when compared with NA. The outcome was whether a woman had been diagnosed as having an SSI during the hospitalization or was re-hospitalized within 30 days after CD for the treatment of SSIs using five or 81 International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes.
Among the 303 834 Taiwanese women who underwent CD during the 5 yr observation period, the 30 day post-CD SSI rate was 0.3% or 0.9% based on five or 81 ICD-9-CM codes. The multivariate-adjusted OR of having post-CD SSIs in the GA group was 3.73 (95% CI, 3.07–4.53) compared with the NA group (P<0.001) using five ICD-9-CM codes for the definition of SSI.
GA for CD was associated with a higher risk of SSI when compared with neuraxial anaesthesia.
PMCID: PMC3243922  PMID: 21857016
anaesthesia; Caesarean section; general anaesthesia; neuraxial anaesthesia; surgical site infection
4.  Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions 
Cell Death and Differentiation  2010;18(3):415-426.
In type II diabetes (T2DM), there is a deficit in β-cells, increased β-cell apoptosis and formation of intracellular membrane-permeant oligomers of islet amyloid polypeptide (IAPP). Human-IAPP (h-IAPP) is an amyloidogenic protein co-expressed with insulin by β-cells. IAPP expression is increased with obesity, the major risk factor for T2DM. In this study we report that increased expression of human-IAPP led to impaired autophagy, due at least in part to the disruption of lysosome-dependant degradation. This action of IAPP to alter lysosomal clearance in vivo depends on its propensity to form toxic oligomers and is independent of the confounding effect of hyperglycemia. We report that the scaffold protein p62 that delivers polyubiquitinated proteins to autophagy may have a protective role against human-IAPP-induced apoptosis, apparently by sequestrating protein targets for degradation. Finally, we found that inhibition of lysosomal degradation increases vulnerability of β-cells to h-IAPP-induced toxicity and, conversely, stimulation of autophagy protects β-cells from h-IAPP-induced apoptosis. Collectively, these data imply an important role for the p62/autophagy/lysosomal degradation system in protection against toxic oligomer-induced apoptosis.
PMCID: PMC3132000  PMID: 20814419
pancreatic β-cell; islet amyloid polypeptide; autophagy; p62/sequestosome 1; type II diabetes mellitus
5.  Integration of CNS survival and differentiation by HIF2α 
Cell Death and Differentiation  2011;18(11):1757-1770.
Hypoxia-inducible factor (HIF) 1α and HIF2α and the inhibitor of apoptosis survivin represent prominent markers of many human cancers. They are also widely expressed in various embryonic tissues, including the central nervous system; however, little is known about their functions in embryos. Here, we show that zebrafish HIF2α protects neural progenitor cells and neural differentiation processes by upregulating the survivin orthologues birc5a and birc5b during embryogenesis. Morpholino-mediated knockdown of hif2α reduced the transcription of birc5a and birc5b, induced p53-independent apoptosis and abrogated neural cell differentiation. Depletion of birc5a and birc5b recaptured the neural development defects that were observed in the hif2α morphants. The phenotypes induced by HIF2α depletion were largely rescued by ectopic birc5a and birc5b mRNAs, indicating that Birc5a and Birc5b act downstream of HIF2α. Chromatin immunoprecipitation assay revealed that HIF2α binds to birc5a and birc5b promoters directly to modulate their transcriptions. Knockdown of hif2α, birc5a or birc5b reduced the expression of the cdk inhibitors p27/cdkn1b and p57/cdkn1c and increased ccnd1/cyclin D1 transcription in the surviving neural progenitor cells. The reduction in elavl3/HuC expression and enhanced pcna, nestin, ascl1b and sox3 expression indicate that the surviving neural progenitor cells in hif2α morphants maintain a high proliferation rate without terminally differentiating. We propose that a subset of developmental defects attributed to HIF2α depletion is due in part to the loss of survivin activity.
PMCID: PMC3190110  PMID: 21546908
HIF2α; surviving; neural progenitor cells; apoptosis
6.  Faecal ribosomal protein L19 is a genetic prognostic factor for survival in colorectal cancer 
Ribosomal proteins are encoded by a gene family, members of which are overexpressed in human cancers. Many of them have been found, using oligonucleotide microarray hybridization, to be differentially expressed in the faeces of patients with various stages of col-orectal cancer (CRC). The gene encoding ribosomal protein L19 (RPL19), a prognostic marker for human prostate cancer, is differentially expressed in CRC patients. Measurement of faecal RPL19 mRNA might improve prognostic prediction for CRC patients. Using quantitative real-time reverse transcription PCR, levels of RPL19 mRNA were detected in samples of colonic tissues from 44 CRC patients, in the faeces of 54 CRC patients and 15 controls, and in 11 colonic cell lines. Seven of 24 patients with late-stage CRC (Dukes' stages C and D) expressed over 2-fold more RPL19 in colonic tumour tissues than in corresponding normal tissues (P= 0.038). The mean faecal RPL19 mRNA levels of late-staged patients were higher than those of controls (P= 0.003) and early-staged patients (P= 0.008). Patients with both high serum levels of carcinoembryonic antigen (CEA; >5 ng/mL) and high-faecal RPL19 mRNA (≥0.0069) had higher risk (odds ratio, 8.0; P= 0.015) and lower overall 48-month survival (33.8 ± 13.7%, P= 0.013). Oligonucleotide microarray hybridization analysis of faecal molecules identified gene transcripts differentially present in faeces. In conclusion, faecal RPL19 expression is associated with advanced tumour stages and addictive to serum CEA in predicting prognosis of CRC patients.
PMCID: PMC4506161  PMID: 18266979
colorectal cancer; oligonucleotide microarray; ribosomal protein L19; overall 48-month survival; prognostic marker
7.  Tomotherapy for neurofibromatosis Type 2: case report and review of the literature 
The British Journal of Radiology  2010;83(988):e074-e078.
Neurofibromatosis 2 (NF2) results in multiple central nervous system tumours. In this case report, the patient has one vestibular schwannoma, one trigeminal schwannoma and two meningiomas developed before the age of 30. Aiming to treat three targets at one fraction with minimal interaction and overlapping doses to normal tissue, the sophisticated equipment of tomotherapy was utilised for frameless stereotaxy; tomotherapy delivered intensity-modulated, rotational radiation therapy using a fan-beam delivery. Daily CT scans with the inbuilt CT scanner were also performed as part of the image-guided radiotherapy. The course of fractionated stereotactic radiotherapy consisted of eight fractions given three times per week with an overall treatment time of 17 days. For the meningioma over left parietal vertex, 4.5 Gy per fraction was given at 36 Gy/8 Fr/17 days. For the meningioma over anterior cerebral falx, 4 Gy per fraction was given at 32 Gy/8 Fr/17 days. For the two schwannomas as one target, 5 Gy per fraction was given at 40 Gy/8 Fr/17 days. The acute effect of the treatment was alopecia and mild headache. Subsequent follow-up confirmed clinical improvement. This is the first reported case of clinical experience with tomotherapy in the management of NF2.
PMCID: PMC3473451  PMID: 20335436
8.  A low frequency of pancreatic islet insulin-expressing cells derived from cord blood stem cell allografts in humans 
Diabetologia  2011;54(5):1066-1074.
We sought to establish if stem cells contained in cord blood cell allografts have the capacity to differentiate into insulin-expressing beta cells in humans.
We studied pancreases obtained at autopsy from individuals (n = 11) who had prior opposite-sex cord blood transplants to reconstitute haematopoiesis. Pancreatic tissue sections were stained first by XY-fluorescence in situ hybridisation and then insulin immunohistochemistry. Pancreases obtained at autopsy from participants without cord blood cell infusions served as controls (n = 11).
In the men with prior transplant of female cord blood, there were 3.4 ± 0.3% XX-positive insulin-expressing islet cells compared with 0.32 ± 0.05% (p < 0.01) in male controls. In women with prior transplant of male cord blood cells we detected 1.03 ± 0.20% XY insulin-expressing islet cells compared with 0.03 ± 0.03 in female controls (p < 0. 001).
Cord blood stem cells have the capacity to differentiate into insulin-expressing cells in non-diabetic humans. It remains to be established whether these cells have the properties of beta cells.
PMCID: PMC3071928  PMID: 21331470
Beta cell; Cord blood cell; Diabetes; Stem cell
9.  Effects of luteolin and quercetin, inhibitors of tyrosine kinase, on cell growth and metastasis-associated properties in A431 cells overexpressing epidermal growth factor receptor 
British Journal of Pharmacology  1999;128(5):999-1010.
Flavonoids display a wide range of pharmacological properties including anti-inflammatory. Anti-mutagenic, anti-carcinogenic and anti-cancer effects. Here, we evaluated the effects of eight flavonoids on the tumour cell proliferation, cellular protein phosphorylation, and matrix metalloproteinase (MMPs) secretion.Of the flavonoids examined, luteolin (Lu) and quercetin (Qu) were the two most potent agents, and significantly inhibited A431 cell proliferation with IC50 values of 19 and 21 μM, respectively.The epidermal growth factor (EGF) (10 nM) promoted growth of A431 cells (+25±4.6%) and mediated epidermal growth factor receptor (EGFR) tyrosine kinase activity and autophosphorylation of EGFR were inhibited by Lu and Qu. At concentration of 20 μM, both Lu and Qu markedly decreased the levels of phosphorylation of A431 cellular proteins, including EGFR.A431 cells treated with Lu or Qu exhibited protuberant cytoplasmic blebs and progressive shrinkage morphology. Lu and Qu also time-dependently induced the appearance of a ladder pattern of DNA fragmentation, and this effect was abolished by EGF treatment.The addition of EGF only marginally diminished the inhibitory effect of luteolin and quercetin on the growth rate of A431 cells, treatment of cellular proteins with EGF and luteolin or quercetin greatly reduced protein phosphorylation, indicating Lu and Qu may act effectively to inhibit a wide range of protein kinases, including EGFR tyrosine kinase.EGF increased the levels of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), while Lu and Qu appeared to suppress the secretion of these two MMPs in A431 cells.Examination of the relationship between the chemical structure and inhibitory effects of eight flavonoids reveal that the double bond between C2 and C3 in ring C and the OH groups on C3′ and C4′ in ring B are critical for the biological activities.This study demonstrates that the inhibitory effects of Lu and Qu, and the stimulatory effects of EGF, on tumour cell proliferation, cellular protein phosphorylation, and MMP secretion may be mediated at least partly through EGFR. This study supports the idea that Lu and Qu may have potential as anti-cancer and anti-metastasis agents.
PMCID: PMC1571723  PMID: 10556937
Epidermal growth factor receptor; protein tyrosine kinase; matrix metalloproteinase; metastasis; luteolin; quercetin
11.  Expression of green fluorescent protein in oligodendrocytes in a time- and level-controllable fashion with a tetracycline-regulated system. 
Molecular Medicine  1999;5(2):129-137.
Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.
PMCID: PMC2230417  PMID: 10203578
12.  The herpes simplex virus type 1 VP5 promoter contains a cis-acting element near the cap site which interacts with a cellular protein. 
Journal of Virology  1996;70(3):1898-1904.
The promoter controlling the expression of the transcript encoding the major herpes simplex virus type 1 capsid protein (VP5, UL19) extends only 60 bases or so from a functional Sp1 site at --48 to include a cis-acting element 3' of the transcript start site. In the present communication, we report the generation of recombinant viruses bearing mutations between --6 and + 8 relative to the cap site in the VP5 promoter controlling expression of a reporter gene. Analysis of the effects of these mutations upon reporter gene expression along with the results of protein binding assays demonstrates that this cap transcription element functionally interacts with a cellular protein of a normal size of 40 kDa. Thus, like the strict late UL38 promoter characterized earlier, the late VP5 promoter has the essential properties of a cellular promoter.
PMCID: PMC190018  PMID: 8627715
13.  Overlapping initiator and TATA box functions in the basal core promoter of hepatitis B virus. 
Journal of Virology  1995;69(6):3647-3657.
The regulation of transcription of the hepatitis B virus core promoter is an important event in the viral life cycle. Two messages, precore and pregenomic RNAs, that are initiated 30 nucleotides apart are produced by the core promoter. Precore RNA encodes nucleocapsid protein and pregenomic RNA core and polymerase. The latter transcript also serves as a template for viral genome replication via reverse transcription. We have previously defined a basal core promoter, which contains four TA-rich sequences (TA1 through TA4) but no canonical TATA element, that can direct transcription of both messages. In this study, we demonstrated that a stretch of 15 nucleotides containing TA4 is sufficient to direct precise initiation of both precore and pregenomic transcripts. This sequence can function as both an initiator and a TATA element. Mutational analysis further revealed that sequences essential for either function are colocalized. The significance of this finding with respect to the basal transcription mechanism and regulation of viral gene expression is discussed.
PMCID: PMC189080  PMID: 7745713
14.  The activity of the pseudorabies virus latency-associated transcript promoter is dependent on its genomic location in herpes simplex virus recombinants as well as on the type of cell infected. 
Journal of Virology  1994;68(3):1972-1976.
As do many other alphaherpesviruses, pseudorabies virus (PRV) transcribes a limited portion of its viral genome in latently infected neurons during latency. The sequence of the PRV latency-associated transcript (LAT) is bounded on its 5' end by a putative promoter region which contains sequence elements similar to those characterized for the herpes simplex virus (HSV) LAT promoter. Using the bacterial beta-galactosidase gene as a reporter, we have assayed PRV LAT promoter activity in the genomic environment in recombinant HSVs. The PRV LAT promoter-beta-galactosidase reporter gene was recombined into the terminal and internal long repeat regions (RL regions), replacing the normal HSV LAT promoter, the cap site, and the first 60 bases of the primary transcript. When recombined into the RL region, appreciable reporter gene expression was observed following infection of two cell lines of neuronal origin; little or no activity was seen with these recombinants following infection of rabbit skin or mouse embryo fibroblasts. No significant expression was seen when the promoter was recombined into the gC locus in the long unique region in any of the cell types utilized. Such results suggest that the PRV latency promoter contains neuronal cell-specific elements and that the HSV RL region provides an appropriate genomic environment for the manifestation of that specificity.
PMCID: PMC236661  PMID: 8107257
15.  Mutational analysis of sequences downstream of the TATA box of the herpes simplex virus type 1 major capsid protein (VP5/UL19) promoter. 
Journal of Virology  1993;67(9):5109-5116.
Transient expression assays with the herpes simplex virus type 1 (HSV-1) promoter/leader controlling the beta gamma (leaky-late) VP5 (UL19) mRNA encoding the major capsid protein showed that no more than 36 to 72 bases of VP5 leader are required for full-level expression. Constructs lacking the viral leader and the transcription initiation site expressed the reporter gene at about 20% of the maximum level. We confirmed this observation by using recombinant viruses in which VP5 promoter/leader deletions controlling the bacterial beta-galactosidase gene were inserted into the nonessential glycoprotein C (UL44) locus of the genome. Sequences within +36 are required for full-level expression, and removal of all leader sequences including the cap site resulted in a 10-fold decrease in reporter mRNA accumulation. The removal of the leader sequence had a measurable effect upon the kinetics of reporter mRNA accumulation, but insertion of the entire VP5 leader and cap site into a construct in which the reporter gene was controlled by the kinetically early (beta) dUTPase (UL50) promoter did not result in any significant change in the kinetics of dUTPase promoter expression. These results suggest that DNA sequences both 5' and 3' of the TATA box are important determinants of the beta gamma kinetics and levels of VP5 mRNA accumulation in the infected cell.
PMCID: PMC237908  PMID: 8394439
16.  Identification and cloning of genes involved in anaerobic sulfite reduction by Salmonella typhimurium. 
Journal of Bacteriology  1990;172(7):4100-4102.
Transposon Tn5 insertions causing anaerobic cysteine auxotrophy were isolated from a Salmonella typhimurium cysI parent (auxotrophic under aerobic but not anaerobic conditions). Insertions in one mutant group appeared to be in cysG. A second group of insertions, designated asr (anaerobic sulfite reduction), were located near map unit 53 on the S. typhimurium chromosome. They did not cause aerobic or anaerobic auxotrophy in a cys1+ background but did prevent dissimilatory sulfite reduction. Plasmids containing asr DNA cloned from wild-type S. typhimurium conferred anaerobic prototrophy and the ability to produce hydrogen sulfide from sulfite on an Escherichia coli cys1 mutant.
PMCID: PMC213397  PMID: 2163396

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