Biomedical subjects

Yiu-Kay Lai

Publications and source records attributed to Yiu-Kay Lai.

16 recordsLinked to original sources

PubMed2007

Signal peptide of eosinophil cationic protein upregulates transforming growth factor-alpha expression in human cells.

Eosinophil cationic protein (ECP) is a major component of eosinophil granule protein that is used as a clinical bio-marker for asthma and allergic inflammatory diseases. Previously, it has been reported that the signal peptide of human ECP (ECPsp) inhibits the cell growth of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris), but not mammalian A431 cells. The inhibitory effect is due to the lack of human signal peptide peptidase (hSPP), a protease located on the endoplasmic reticulum (ER) membrane, in the lower organisms. In this study, we show that the epidermal growth factor receptor (EGFR) is upregulated by the exogenous ECPsp-eGFP as a result of the increased expression of the transforming growth factor-alpha (TGF-alpha) at both transcriptional and translational levels in A431 and HL-60 clone 15 cell lines. Furthermore, the N-terminus of ECPsp fragment generated by the cleavage of hSPP (ECPspM1-G17) gives rise to over threefold increase of TGF-alpha protein expression, whereas another ECPsp fragment (ECPspL18-A27) and the hSPP-resistant ECPsp (ECPspG17L) do not show similar effect. Our results indicate that the ECPspM1-G17 plays a crucial role in the upregulation of TGF-alpha, suggesting that the ECPsp not only directs the secretion of mature ECP, but also involves in the autocrine system.

Aspartic Acid Endopeptidases↗

PubMed2007

Two non-homologous brain diseases-related genes, SERPINI1 and PDCD10, are tightly linked by an asymmetric bidirectional promoter in an evolutionarily conserved manner.

BACKGROUND: Despite of the fact that mammalian genomes are far more spacious than prokaryotic genomes, recent nucleotide sequencing data have revealed that many mammalian genes are arranged in a head-to-head orientation and separated by a small intergenic sequence. Extensive studies on some of these neighboring genes, in particular homologous gene pairs, have shown that these genes are often co-expressed in a symmetric manner and regulated by a shared promoter region. Here we report the identification of two non-homologous brain disease-related genes, with one coding for a serine protease inhibitor (SERPINI1) and the other for a programmed cell death-related gene (PDCD10), being tightly linked together by an asymmetric bidirectional promoter in an evolutionarily conserved fashion. This asymmetric bidirectional promoter, in cooperation with some cis-acting elements, is responsible for the co-regulation of the gene expression pattern as well as the tissue specificity of SERPINI1 and PDCD10. RESULTS: While SERPINI1 is predominantly expressed in normal brain and down-regulated in brain tumors, PDCD10 is ubiquitously expressed in all normal tissues but its gene transcription becomes aberrant in different types of cancers. By measuring the luciferase activity in various cell lysates, their 851-bp intergenic sequence was shown to be capable of driving the reporter gene expression in either direction. A 175-bp fragment from nt 1 to 175 in the vicinity of PDCD10 was further determined to function as a minimal bidirectional promoter. A critical regulatory fragment, from nt 176-473 outside the minimal promoter in the intergenic region, was identified to contain a strong repressive element for SERPINI1 and an enhancer for PDCD10. These cis-acting elements may exist to help coordinate the expression and regulation of the two flanking genes. CONCLUSION: For all non-homologous genes that have been described to be closely adjacent in the mammalian genomes, the intergenic region of the head-to-head PDCD10-SERPINI1 gene pair provides an interesting and informative example of a complex regulatory system that governs the expression of both genes not only through an asymmetric bidirectional promoter, but also through fine-tuned regulations with some cis-acting elements.

Apoptosis Regulatory Proteins↗

PubMed2006

Clinical evaluation of the eBsensor hand-held blood glucose monitoring system.

BACKGROUND: Home glucose monitoring system is increasingly recognized as an important tool for glycemic control. We evaluated the clinical performance of the eBsensor glucose monitoring system. METHODS: Fingertip capillary blood glucose concentrations from 282 subjects were measured using eBsensor glucose monitoring system and compared against predicate devices and the Yellow Springs Instruments (YSI) 2300 blood glucose analyzer. Accuracy and precision of the eBsensor glucose monitoring system were assessed using several methods. The comparative study between the eBsensor and 2 currently marketed monitoring systems was performed. RESULTS: The 282 eBsensor readings covered a wide range from 2.6 to 24.4 mmol/l. Deming regression and Pearson correlation analyses showed a linear relationship between the eBsensor readings and the YSI reference method (eBsensor=0.9496 YSI+0.4127 mmol/l; r=0.98). Error Grid analysis demonstrated that 100% of the eBsensor readings in clinically acceptable zones A and B. The CVs for the 6 lots of strips were within the satisfactory interval (<6%). The comparative study showed that the eBsensor readings correlated well with the OneTouch Ultra values (r=0.97) and the Glucocard II values (r=0.97). CONCLUSIONS: eBsensor is a reliable glucose monitoring system which provides high accurate and precise glucose readings over a wide range of glucose concentrations.

Blood Glucose Self-Monitoring↗

PubMed2006

Inhibition of hepatitis B virus production by Boehmeria nivea root extract in HepG2 2.2.15 cells.

AIM: To explore the anti-hepatitis B virus (HBV) effects of Boehmeria nivea (B. nivea) root extract (BNE) by using the HepG2 2.2.15 cell model system. METHODS: Hepatitis B surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), and HBV DNA were measured by using ELISA and real-time PCR, respectively. Viral DNA replication and RNA expression were determined by using Southern and Northern blot, respectively. RESULTS: In HepG2 2.2.15 cells, HBeAg (60%, P < 0.01) and particle-associated HBV DNA (> 99%, P < 0.01) secretion into supernatant were significantly inhibited by BNE at a dose of 100 mg/L, whereas the HBsAg was not inhibited. With different doses of BNE, the reduced HBeAg was correlated with the inhibition of HBV DNA. The anti-HBV effect of BNE was not caused by its cytotoxicity to cells or inhibition of viral DNA replication and RNA expression. CONCLUSION: BNE could effectively reduce the HBV production and its anti-HBV machinery might differ from the nucleoside analogues.

Antiviral Agents↗

PubMed2006

Accelerated induction of apoptosis in insect cells by baculovirus-expressed SARS-CoV membrane protein.

It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed.

Animals↗

PubMed2006

Differential expression of Hsp90 isoforms in geldanamycin-treated 9L cells.

In mammals, two major Hsp90 isoforms (Hsp90alpha and Hsp90beta) have been identified and found to be highly conserved among different species. However, the expression control of Hsp90 isoforms at both transcriptional and translational levels is largely unknown. Herein, we quantitatively investigate the changes in the total mRNA and inductive protein levels of Hsp90alpha and Hsp90beta in rat gliosarcoma cells treated with geldanamycin (GA). The stability of mRNA and protein was estimated. The translational efficiency of Hsp90 isoforms was measured employing in vitro translation techniques. It was found that Hsp90alpha was more inducible than Hsp90beta after GA treatment, whereas the hsp90alpha mRNA level was lower than that of hsp90beta. In addition, higher translational efficiency of hsp90alpha mRNA was observed, suggesting that translational control played an important role. Taken together, our results indicate that differential expression between Hsp90alpha and Hsp90beta is a consequence of both distinct mRNA profiles and differential translation processes.

Animals↗

PubMed2006

Localization of GRP78 to mitochondria under the unfolded protein response.

The ubiquitously expressed molecular chaperone GRP78 (78 kDa glucose-regulated protein) generally localizes to the ER (endoplasmic reticulum). GRP78 is specifically induced in cells under the UPR (unfolded protein response), which can be elicited by treatments with calcium ionophore A23187 and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor TG (thapsigargin). By using confocal microscopy, we have demonstrated that GRP78 was concentrated in the perinuclear region and co-localized with the ER marker proteins, calnexin and PDI (protein disulphide-isomerase), in cells under normal growth conditions. However, treatments with A23187 and TG led to diminish its ER targeting, resulting in redirection into a cytoplasmic vesicular pattern, and overlapping with the mitochondrial marker MitoTracker. Cellular fractionation and protease digestion of isolated mitochondria from ER-stressed cells suggested that a significant portion of GRP78 is localized to the mitochondria and is protease-resistant. Localizations of GRP78 in ER and mitochondria were confirmed by using immunoelectron microscopy. In ER-stressed cells, GRP78 mainly localized within the mitochondria and decorated the mitochondrial membrane compartment. Submitochondrial fractionation studies indicated further that the mitochondria-resided GRP78 is mainly located in the intermembrane space, inner membrane and matrix, but is not associated with the outer membrane. Furthermore, radioactive labelling followed by subcellular fractionation showed that a significant portion of the newly synthesized GRP78 is localized to the mitochondria in cells under UPR. Taken together, our results indicate that, at least under certain circumstances, the ER-resided chaperone GRP78 can be retargeted to mitochondria and thereby may be involved in correlating UPR signalling between these two organelles.

Animals↗

PubMed2006

Involvement of calcium in the differential induction of heat shock protein 70 by heat shock protein 90 inhibitors, geldanamycin and radicicol, in human non-small cell lung cancer H460 cells.

Both geldanamycin (GA) and radicicol (RA) are HSP90 binding agents that possess antitumour activities. Although the in vitro data indicated that the inhibitory constant of RA is much bigger than that of GA, the in vivo data on drug efficacy might reveal different results. We have recently shown that treatment with GA induces a heat-shock response and that calcium mobilization may be involved in the process. By using induction of HSP70 as the endpoint assay, we found changes in upstream signaling mediators, including HSF1 and calcium mobilization, as well as possible involvement of protein kinase in human non-small cell lung cancer H460 cells treated with GA and RA. Our results demonstrated that calcium mobilization, a calcium dependent and H7-sensitive protein kinase, along with HSF1 activation by phosphorylation, are all involved in the HSP70 induction process triggered by the drugs. However, only GA, but not RA, can provoke a rapid calcium mobilization and thereby result in an instant induction of HSP70. Furthermore, the rapid calcium influx, followed by instant HSP induction, could be achieved in GA- or RA-treated cells placed in a medium containing excessive calcium while the response was completely abolished in cells depleted of calcium. Taken together, our findings suggest that differential calcium signaling may account for the differential induction of HSP and the action of GA and RA.

Benzoquinones↗

PubMed2006

Stable RNA interference in Spodoptera frugiperda cells by a DNA vector-based method.

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study.

Animals↗

PubMed2006

Combining multiplex reverse transcription-PCR and a diagnostic microarray to detect and differentiate enterovirus 71 and coxsackievirus A16.

Cluster A enteroviruses, including enterovirus 71 (EV71) and coxsackievirus A16 (CA16), are known to cause hand-foot-and-mouth disease (HFMD). Despite the close genetic relationship between these two viruses, EV71 is generally known to be a more perpetuating pathogen involved in severe clinical manifestations and deaths. While the serotyping of enteroviruses is mostly done by conventional immunological methods, many clinical isolates remain unclassifiable due to the limited number of antibodies against enterovirus surface proteins. Array-based assays are able to detect several serotypes with high accuracy. We combined an enterovirus microarray with multiplex reverse transcription-PCR to try to develop a method of sensitively and accurately detecting and differentiating EV71 and CA16. In an effort to design serotype-specific probes for detection of the virus, we first did an elaborate bioinformatic analysis of the sequence database derived from different enterovirus serotypes. We then constructed a microarray using 60-mer degenerate oligonucleotide probes covalently bound to array slides. Using this enterovirus microarray to study 144 clinical specimens from patients infected with HFMD or suspected to have HFMD, we found that it had a diagnostic accuracy of 92.0% for EV71 and 95.8% for CA16. Diagnostic accuracy for other enteroviruses (non-EV71 or -CA16) was 92.0%. All specimens were analyzed in parallel by real-time PCR and subsequently confirmed by neutralization tests. This highly sensitive array-based assay may become a useful alternative in clinical diagnostics of EV71 and CA16.

DNA Probes↗

PubMed2005

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C.

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor Gö-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.

Amino Acid Sequence↗

PubMed2005

Expression and purification of N and E proteins from severe acute respiratory syndrome (SARS)-associated coronavirus: a comparative study.

Histidine-tagged N (rNH) and E (rEH) proteins of Severe Acute Respiratory Syndrome (SARS)-coronovirus were expressed in the baculovirus/insect cell system and purified by immobilized metal affinity chromatography. rNH and rEH proteins differed markedly with respect to expression levels, cell death kinetics and subcellular localizations that led to different extraction and purification schemes. The features of both proteins are compared and the potential applications of purified rNH and rEH are discussed.

Animals↗

PubMed2004

Coupling multiplex RT-PCR to a gene chip assay for sensitive and semiquantitative detection of severe acute respiratory syndrome-coronavirus.

An early and accurate diagnostic assay for severe acute respiratory syndrome (SARS) is crucial for infection control. However, most of the diagnostic methods available today, such as real-time reverse transcriptase-polymerase chain reaction (RT-PCR), require a second detection method for confirmation because they detect a single sequence region of the SARS-coronavirus (SARS-CoV). For sensitive and accurate early diagnosis, we report a novel assay system combining multiplex RT-PCR and a diagnostic gene chip to detect multiple virus-specific genomic sequences of SARS-CoV. With 53 clinical specimens, we successfully demonstrate that this technique offers not only a high-accuracy diagnosis for early SARS infection but also a semiquantitative assay.

DNA Primers↗

PubMed2003

Retaining of the assembly capability of vimentin phosphorylated by mitogen-activated protein kinase-activated protein kinase-2.

Intermediate filament (IF) networks can be regulated by phosphorylation of unit proteins, such as vimentin, by specific kinases leading to reorganization of the IF filamentous structure. Recently, we identified mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) as a vimentin kinase (Cheng and Lai [1998] J. Cell. Biochem. 71:169-181). Herein we describe the results of further in vitro studies investigating the effects of MAPKAP kinase-2 phosphorylation on vimentin and the effects of the phosphorylation on the filamentous structure. We show that MAPKAP kinase-2 mainly phosphorylates vimentin at Ser-38, Ser-50, Ser-55, and Ser-82, residues all located in the head domain of the protein. Surprisingly, and in stark contrast to phosphorylation by most other kinases, phosphorylation of vimentin by MAPKAP kinase-2 has no discernable effect on its assembly. It suggested that structure disassembly is not the only obligated consequence of phosphorylated vimentin as regulated by other kinases. Finally, a mutational analysis of each of the phosphorylated serine residues in vimentin suggested that no single serine site was primarily responsible for structure maintenance, implying that the retention of filamentous structure may be the result of the coordinated action of several phosphorylated serine sites. This also shed new lights on the functional task(s) of vimentin that is intermediate filament proteins might provide a phosphate reservoir to accommodate the phosphate surge without any structural changes.

Base Sequence↗

PubMed2003

Mitochondrial calcium-mediated reactive oxygen species are essential for the rapid induction of the grp78 gene in 9L rat brain tumour cells.

The glucose-regulated protein grp78 gene is rapidly transactivated in 9L rat brain tumour (RBT) cells treated with okadaic acid (OA) followed by heat shock (HS) (termed OA-->HS treatment). By Northern blotting analyses and transient transfection assays, we herein show that transactivation of grp78 by OA-->HS is abolished by an intracellular calcium chelator, bis(aminophenoxy)ethane N,N'-tetraacetic acid (BAPTA), and an inhibitor of mitochondrial Ca(2+) uniporter, ruthenium red (RR), while unaffected by cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MTP). The inhibitory effects of BAPTA and RR also present in OA-->HS induction of transient elevation of intracellular hydrogen peroxide. The requirement of reactive oxygen intermediates (ROIs) is confirmed by substitutional addition of antioxidants, N-acetyl cysteine (NAC) and pyrrolidinedithiocarbamate (PDTC) during OA-->HS treatment, mimicking these inhibitory effects of BAPTA and RR. Western blotting analyses show that phosphorylation of transcription factor CREB is diminished only by BAPTA but not by RR, while phosphorylation of ATF-2 is unaffected by either agent. Conclusively, we present that both the disturbances of mitochondrial calcium homeostasis and reactive oxygen intermediates are essential for rapid transactivation of grp78, and this pathway is separate from protein kinase A (PKA)-dependent CREB activation or p38 mitogen-activated protein kinase (p38(MAPK))-dependent ATF-2 activation and signalling.

Activating Transcription Factor 2↗

PubMed2003

Geldanamycin induction of grp78 requires activation of reactive oxygen species via ER stress responsive elements in 9L rat brain tumour cells.

The molecular mechanism whereby anticancer agent geldanamycin (GA) impacts endoplasmic reticulum (ER) stress pathway is largely unknown. Here, we investigate the effect of GA on the expression of grp78 coding for ER stress protein and the mechanistic relationship of GA signalling to ER stress. GA induces the expression of mRNA and protein of grp78 by Northern blot analysis and metabolic labelling experiment in cultured rat brain tumour 9L cells. The induced grp78 expression is sensitive to antioxidant N-acetylcysteine (NAC) addition, indicating the involvement of reactive oxygen species (ROS) in GA-induced ER stress. Results from direct determination of oxidation status using dichlorodihydrofluorescein diacetate (H(2)DCFDA) showed that accumulation of ROS elicited GA was quenched by addition of NAC. Reporter genes harbouring deletions of transcription elements from grp78 promoter demonstrated that controlling elements of ERSE1, ERSE2 and CRE are required in GA treatment. The critical ROS-dependent elements in grp78 promoter can be confined within ER stress responsive element (ERSE) region, since reporter constructs loss of ERSE elements that lost the susceptibility to be modulated by NAC after GA treatment. Hence, ER stress elements correlate well with ROS-mediated elements in grp78 promoter. Reporter construct loss of ERSE element retains the susceptibility by NAC after GA treatment, indicating that CRE element might represent a ROS-independent, GA-inductive element. Conclusively, we show that ROS is required for GA to launch the transactivation of grp78, and a firm link was established between the ROS signalling pathway to specific promoter elements-ERSE1 and ERSE2 elements in ER stress marker gene grp78 promoter.

Acetylcysteine↗