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Biomedical subjects

A L Cheung

Publications and source records attributed to A L Cheung.

At least 19 recordsLinked to original sources
PubMed2005

The detection of small relative simulated field defects using multifocal VEPs.

INTRODUCTION: The multifocal visual-evoked potential (mfVEP) has been widely used in the study of diseases of the visual system. However, the sensitivity of the mfVEP in the objective detection of relative field defects has not been determined. This study investigates variations in mfVEP responses while simulating relative field defects by using different luminous transmission masks [neutral density (ND) filters] on the stimulus pattern. METHODS: Simulated relative field defects with four different luminous transmissions were obtained by using 0.2, 0.4, 0.6, and 0.8 ND filters, 5 degrees in size, at two different retinal eccentricities (10 and 16 degrees) on a standard mfVEP dartboard stimulus. Eleven normal subjects were recruited for mfVEP measurements. The response amplitudes and latencies of the N1 and P1 of the mfVEP, with and without small simulated relative field defects, were compared. RESULTS: The mfVEP amplitudes of N1 and P1 decreased substantially when 0.6 and 0.8 ND filters were introduced. The effects were similar at both the 10- and 16-degree eccentricities but there was no change in latency with simulated field defects at either location. CONCLUSIONS: The mfVEP can detect a simulated relative field defect 5 degrees in size starting with 0.6 log unit reduction in luminance at both 10-degree and 16-degree eccentricities. This illustrates that the sensitivity of the mfVEP measurement is nearly comparable with that of the Humphrey Visual Field Analyser.

Adult↗
PubMed2003

Characterization of RAT, an autolysis regulator in Staphylococcus aureus.

In trying to identify genetic loci involved in the regulation of cap5 genes in Staphylococcus aureus, we isolated a transposon mutant that exhibited a growth defect, enhanced autolysis and increased sensitivity to Triton X-100 and penicillin, attributable in part to increased murein hydrolase activity. Analysis of the chromosomal sequence flanking the transposon insertion site revealed that the gene disrupted in the mutant encodes an open reading frame of 147 amino acids. We named this gene rat, which stands for regulator of autolytic activity. Sequence analysis indicated that Rat is homologous to the MarR and, to a lesser extent, the SarA protein families. Mutations in rat resulted in decreased expression of known autolytic regulators lytSR, lrgAB and arlRS. Gel shift studies indicated that Rat binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. As anticipated, rat appears to be a negative regulator of autolysin genes including lytM and lytN. These data suggest that the rat gene product is an important regulator of autolytic activity in S. aureus.

Amino Acid Sequence↗
PubMed2001

Effect of p53 on centrosome amplification in prostate cancer cells.

Chromosomal instability (CIN) is one of the common features in prostate cancer, especially in advanced stages. Recently, the involvement of p53 in CIN through the regulation of centrosome amplification has been proposed in certain tumor types. In this study, we investigated the relationship between p53 and centrosome amplification in prostate cancer cells. Increased centrosome number and size were observed in DU145 and PC3 containing nonfunctional p53 compared to LNCap which expressed wild-type p53. Transfection of p53 into PC3 cells resulted in a decreased cell growth rate, G2/M arrest and decreased centrosome abnormalities. We provide the first evidence on a correlation between loss of p53 function and centrosome amplification in prostate cancer cells. Our results indicate that p53 may play a role in the regulation of centrosome amplification and loss of p53 may be one of the mechanisms involving CIN in prostate cancer cells.

Centrosome↗
PubMed2001

E-cadherin expression is commonly downregulated by CpG island hypermethylation in esophageal carcinoma cells.

E-cadherin, a cell adhesion molecule, is regarded as an invasion-suppressor molecule and a prognostic marker in many types of human cancers. Downregulation of E-cadherin is common in esophageal carcinoma and is associated with an increase in invasive and metastatic potential. To study the mechanisms responsible for inactivation of this gene in esophageal squamous cell carcinoma (ESCC), we investigated the methylation status around the 5' promoter region of E-cadherin gene of six ESCC cell lines by methylation-specific polymerase chain reaction, and compared it with E-cadherin protein and mRNA expression. We also studied the methylation status of 20 ESCC clinical specimens. Methylation was noted in four of the six cell lines (one fully methylated and three partially methylated). The completely methylated cell line lacked E-cadherin protein expression and mRNA transcription. E-cadherin expression and transcription were reduced in a partially methylated cell line but preserved in the other partially methylated cell lines. Treatment of E-cadherin-negative carcinoma cells with the demethylating agent, 5-aza-2'-deoxycytidine, induced re-expression of the gene. A high frequency of methylation (16/20, 80%) was also noted in the 20 ESCC clinical samples. Our results indicate that 5' CpG island methylation is common in esophageal carcinoma and may play an important role in downregulation of E-cadherin.

Azacitidine↗
PubMed2001

Crystal structure of the SarR protein from Staphylococcus aureus.

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). The sar (Staphylococcus accessory regulator) locus is composed of three overlapping transcripts (sarA P1, P3, and P2, transcripts initiated from the P1, P3, and P2 promoters, respectively), all encoding the 124-aa SarA protein. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of the sarA promoters. We previously partially purified a 13.6-kDa protein, designated SarR, that binds to the sarA promoter region to down-modulate sarA transcription from the P1 promoter and subsequently SarA expression. SarR shares sequence similarity to SarA, and another SarA homolog, SarS. Here we report the 2.3 A-resolution x-ray crystal structure of the dimeric SarR-MBP (maltose binding protein) fusion protein. The structure reveals that the SarR protein not only has a classic helix-turn-helix module for DNA binding at the major grooves, but also has an additional loop region involved in DNA recognition at the minor grooves. This interaction mode could represent a new functional class of the "winged helix" family. The dimeric SarR structure could accommodate an unusually long stretch of approximately 27 nucleotides with two or four bending points along the course, which could lead to the bending of DNA by 90 degrees or more, similar to that seen in the catabolite activator protein (CAP)-DNA complex. The structure also demonstrates the molecular basis for the stable dimerization of the SarR monomers and possible motifs for interaction with other proteins.

Amino Acid Sequence↗
PubMed2001

Downregulation of hemidesmosomal proteins in nasopharyngeal carcinoma cells.

Hemidesmosome (HD) is a transmembrane complex that mediates attachment of epithelial cells to the basement membrane. Abnormal expression of HD components has been reported in several types of human cancers and is believed to play a role in tumor invasion and metastasis. Using differential gene display, we have identified downregulation of BPAG1 expression in nasopharyngeal carcinoma cells. BPAG1 is a major component of hemidesmosome. In the present study, we have extended our work to investigate the expression pattern of other components in the HD complex, namely, BPAG2, ITGalpha6 and ITGbeta4 in three distinct biological groups of nasopharyngeal epithelial cells: (a) non-malignant nasopharyngeal epithelial cells established from primary culture of nasopharyngeal explants, (b) non-malignant nasopharyngeal epithelial cells immortalized by viral oncogenes, SV40 or HPV16E6E7, and (c) nasopharyngeal carcinoma (NPC) cells. Both non-malignant primary cultured nasopharyngeal epithelial cells and immortalized nasopharyngeal epithelial cell lines expressed all the HD components examined, although the immortalized cells expressed a lower level of HD components compared with the non-malignant nasopharyngeal cells established from primary culture. In contrast, downregulation of HD components is commonly observed in nasopharyngeal carcinoma cells. Loss of HD expression in NPC may be associated with the undifferentiated properties of NPC cells and may have prognostic significance.

Antigens, CD↗
PubMed2001

Evaluation of a tetracycline-inducible promoter in Staphylococcus aureus in vitro and in vivo and its application in demonstrating the role of sigB in microcolony formation.

An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpac-lacI system, and the arabinose-inducible P(BAD) promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetO promoter::gfp(uvr) fusion carried on a shuttle plasmid, we demonstrated that dose-dependent tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the sigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.

Animals↗
PubMed2001

sigma(B) activity in Staphylococcus aureus is controlled by RsbU and an additional factor(s) during bacterial growth.

Two genes of the sigB operon, rsbU and rsbV, were deleted in an rsbU(+) strain (FDA486) to evaluate the contribution of these two genes to sigma(B) activity in Staphylococcus aureus. The sigma(B) protein level and the transcription of two sigma(B)-dependent promoters (sigB and sarA P3 transcripts) were analyzed in the constructed mutants. A deletion of the first gene (rsbU) within the sigB operon led only to a partial reduction in sigma(beta) activity. A deletion of the second gene (rsbV) resulted in a more dramatic reduction in the sigma(B) protein level and its activity than did the deletion of rsbU, thus indicating that RsbV can be activated independent of RsbU. In the parental strain, the sigma(B)-dependent transcript initiated upstream of rsbV was 28-fold higher than the sigma(A)-dependent transcript originating from the rsbU promoter. The level of the sigma(B)-dependent transcript decreased up to 50% in the rsbU mutant and up to 90% in the rsbV mutant compared with the transcript in the wild type. The yellow pigment of S. aureus colonies, a sigma(B)-dependent phenotype, was partially reduced in the rsbU and rsbV mutants, whereas alpha-hemolysin was increased. Additionally, the sarA P3 promoter activity of the parental strain was induced to a higher level in response to pH 5.5 than was that of the rsbU or rsbV mutant, indicating that RsbU is the major activator of the sigma(B) response to acid stress. Using a tetracycline-inducible system to modulate the expression of RsbW, we progressively repressed pigment production, presumably by reducing the free sigma(B) level. Collectively, our data indicated that RsbU and RsbV in S. aureus contributed to different levels of sigma(B) protein expression and varying sigma(B) activities. Although RsbV can activate sigma(B) independent of RsbU, RsbU remains the major activator of sigma(B) during acid stress.

Bacterial Proteins↗
PubMed2001

Characterization of sarR, a modulator of sar expression in Staphylococcus aureus.

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sar and agr). The sar locus is composed of three overlapping transcripts (sar P1, P3, and P2 transcripts from P1, P3, and P2 promoters, respectively), all encoding the 372-bp sarA gene. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of sar promoters. We previously partially purified a approximately 12 kDa protein with a DNA-specific column containing a sar P2 promoter fragment. In this study, the putative gene, designated sarR, was identified and found to encode a 13.6-kDa protein with homology to SarA. Transcriptional and immunoblot studies revealed the sarR gene to be expressed in other staphylococcal strains. Recombinant SarR protein bound sar P1, P2, and P3 promoter fragments in gel shift and footprinting assays. A sarR mutant expressed a higher level of P1 transcript than the parent, as confirmed by promoter green fluorescent protein fusion assays. As the P1 transcript is the predominant sar transcript, we confirmed that the sarR mutant expressed more SarA than the parental strain. We thus proposed that SarR is a regulatory protein that binds to the sar promoters to down-regulate P1 transcription and the ensuing SarA protein expression.

Amino Acid Sequence↗
PubMed2001

SarS, a SarA homolog repressible by agr, is an activator of protein A synthesis in Staphylococcus aureus.

The expression of protein A (spa) is repressed by global regulatory loci sarA and agr. Although SarA may directly bind to the spa promoter to downregulate spa expression, the mechanism by which agr represses spa expression is not clearly understood. In searching for SarA homologs in the partially released genome, we found a SarA homolog, encoding a 250-amino-acid protein designated SarS, upstream of the spa gene. The expression of sarS was almost undetectable in parental strain RN6390 but was highly expressed in agr and sarA mutants, strains normally expressing high level of protein A. Interestingly, protein A expression was decreased in a sarS mutant as detected in an immunoblot but returned to near-parental levels in a complemented sarS mutant. Transcriptional fusion studies with a 158- and a 491-bp spa promoter fragment linked to the xylE reporter gene disclosed that the transcription of the spa promoter was also downregulated in the sarS mutant compared with the parental strain. Interestingly, the enhancement in spa expression in an agr mutant returned to a near-parental level in the agr sarS double mutant but not in the sarA sarS double mutant. Correlating with this divergent finding is the observation that enhanced sarS expression in an agr mutant was repressed by the sarA locus supplied in trans but not in a sarA mutant expressing RNAIII from a plasmid. Gel shift studies also revealed the specific binding of SarS to the 158-bp spa promoter. Taken together, these data indicated that the agr locus probably mediates spa repression by suppressing the transcription of sarS, an activator of spa expression. However, the pathway by which the sarA locus downregulates spa expression is sarS independent.

Amino Acid Sequence↗
PubMed2001

Clumping factor A mediates binding of Staphylococcus aureus to human platelets.

The direct binding of bacteria to platelets may be an important virulence mechanism in the pathogenesis of infective endocarditis. We have previously described Staphylococcus aureus strain PS12, a Tn551-derived mutant of strain ISP479, with reduced ability to bind human platelets in vitro. When tested in an animal model of endocarditis, the PS12 strain was less virulent than its parental strain, as measured by bacterial densities in endocardial vegetations and incidence of systemic embolization. We have now characterized the gene disrupted in PS12 and its function in platelet binding. DNA sequencing, Southern blotting, and PCR analysis indicate that PS12 contained two Tn551 insertions within the clumping factor A (ClfA) locus (clfA). The first copy was upstream from the clfA start codon and appeared to have no effect on ClfA production. The second insertion was within the region encoding the serine aspartate repeat of ClfA and resulted in the production of a truncated ClfA protein that was secreted from the cell. A purified, recombinant form of the ClfA A region, encompassing amino acids 40 through 559, significantly reduced the binding of ISP479C to human platelets by 44% (P = 0.0001). Immunoprecipitation of recombinant ClfA that had been incubated with solubilized platelet membranes coprecipitated a 118-kDa platelet membrane protein. This protein does not appear to be glycoprotein IIb. These results indicate that platelet binding by S. aureus is mediated in part by the direct binding of ClfA to a novel 118-kDa platelet membrane receptor.

Bacterial Adhesion↗
PubMed2001

Adherence properties of Staphylococcus aureus under static and flow conditions: roles of agr and sar loci, platelets, and plasma ligands.

Global regulatory genes in Staphylococcus aureus, including agr and sar, are known to regulate the expression of multiple virulence factors, including cell wall adhesins. In the present study, the adherence of S. aureus RN6390 (wild type), RN6911 (agr), ALC136 (sar), and ALC135 (agr sar) to immobilized fibrinogen, fibronectin, von Willebrand factor (vWF), extracellular matrix (ECM), and human endothelial cells (EC) EAhy.926 was studied. Bacteria grown to postexponential phase were subjected to light oscillation (static condition) or to shear stress at 200 s(-1) (flow condition) on tissue culture polystyrene plates coated with either protein ligands, ECM, or EC. Adherence of nonlabeled bacteria to immobilized ligands was measured by an image analysis system, while adherence of [(3)H]thymidine-labeled S. aureus to ECM and EC was measured by a beta-scintillation counter. The results showed increased adherence of agr and agr sar mutants to immobilized fibrinogen and higher potential of these mutants to induce platelet aggregation in suspension, decreased adherence of sar and agr sar mutants to immobilized fibronectin and vWF as well as to ECM and EC, increased adherence of both S. aureus wild type and sar mutant to EC treated with platelet-rich plasma (PRP) compared to platelet-poor plasma (PPP) and to EC treated with PPP compared to the control, and increased adherence of S. aureus wild type to EC coated with PRP in which platelets were activated with phorbol 12-myristate 13-acetate compared to intact PRP. This finding paralleled the increased adherence to EC of activated compared to intact platelets. It is suggested that platelet-mediated S. aureus adherence to EC depends on platelet activation and the number of adherent platelets and available receptors on the platelet membrane. In conclusion, the agr locus downregulates S. aureus adherence to fibrinogen, while the sar locus upregulates S. aureus adherence to fibronectin, vWF, ECM, and EC. The effect of both agr and sar on S. aureus adherence properties develops primarily under flow conditions, which suggests different adhesion mechanisms in static and flow conditions.

Animals↗
PubMed2001

SarT, a repressor of alpha-hemolysin in Staphylococcus aureus.

In searching the Staphylococcus aureus genome, we found several homologs to SarA. One of these genes, sarT, codes for a basic protein with 118 residues and a predicted molecular size of 16,096 Da. Northern blot analysis revealed that the expression of sarT was repressed by sarA and agr. An insertion sarT mutant generated in S. aureus RN6390 and 8325-4 backgrounds revealed minimal effect on the expression of sarR and sarA. The RNAIII level was notably increased in the sarT mutant, particularly in postexponential-phase cells, while the augmentative effect on RNAII was less. SarT repressed the expression of alpha-hemolysin, as determined by Northern blotting, Western blotting, and a rabbit erythrocyte hemolytic assay. This repression was relieved upon complementation. Similar to agr and sarA mutants, which predictably displayed a reduction in hla expression, the agr sarT mutant exhibited a lower level of hla transcription than the sarT mutant. In contrast, hla transcription was enhanced in the sarA sarT mutant compared with the single sarA mutant. Collectively, these results indicated that the sarA locus, contrary to the regulatory action of agr, induced alpha-hemolysin production by repressing sarT, a repressor of hla transcription.

Amino Acid Sequence↗
PubMed2000

Effects of neurotrophic factors on motoneuron survival following axonal injury in newborn rats.

Using two different lesion models, the spinal root avulsion and the distal nerve axotomy, the present study investigated effects of known neurotrophic factors on motoneuron survival in newborn rats. Results of the present study show that 100% of motoneurons in the lesioned spinal segment die at 1 week following root avulsion, and more than 80% of them die at 2 weeks following distal nerve axotomy. Local application of GDNF can rescue 92% of motoneurons up to 1 week from degeneration due to root avulsion and almost 100% of them up to 2 weeks from degeneration due to distal nerve axotomy. Local application of BDNF fails to prevent any motoneuron death in newborn rats following root avulsion, but it can rescue about 50% of motoneurons up to 2 weeks from degeneration due to distal nerve axotomy. CNTF and IGF-1 fail to prevent any motoneuron death following either distal nerve axotomy or root avulsion. Thus, comparing all the neurotrophic factors tested in this study, GDNF is most effective in preventing death of motoneurons following axonal injury in newborn rats.

Animals↗
PubMed2000

Survival of Staphylococcus aureus inside neutrophils contributes to infection.

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar-) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.

Animals↗
PubMed2000

Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) occurs with a high incidence in many countries in south-eastern Asia. Chromosomal abnormalities have been commonly found in NPC, but the underlying mechanism is not well understood. We determined mitotic indices, the staining pattern of nuclear DNA and cell cycle profiles of NPC cells in response to treatment with microtubule-disrupting agents, and found that the mitotic checkpoint was defective in two out of five (40%) of the tested NPC cell lines. We also observed that an aberrantly reduced expression of MAD2, one of the key components of mitotic checkpoint, correlated with the loss of checkpoint control. Our findings suggest that a defective mitotic checkpoint characterized by reduced expression of MAD2 contributes to chromosomal instability in NPC.

Asia, Southeastern↗
PubMed2000

Staphylococcus aureus RN6390 replicates and induces apoptosis in a pulmonary epithelial cell line.

Staphylococcus aureus frequently colonizes the airways of patients with compromised airway defenses (e.g., cystic fibrosis [CF] patients) for extended periods. Persistent and relapsing infections may be related to live S. aureus bacteria actively residing inside epithelial cells. In this study, we infected a respiratory epithelial cell line, which was derived from a CF patient, with S. aureus RN6390. Internalization of S. aureus was found to be time and dose dependent and could be blocked by cytochalasin D. Transmission electron microscopy revealed that internalized bacteria resided within endocytic vacuoles without any evidence of lysosomal fusion in a 24-h period. The results of internalization experiments and time-lapse fluorescence microscopy of epithelial cells infected with green fluorescent S. aureus indicate that, after an initial lag period of 7 to 9 h, intracellular bacteria began to replicate, with three to five divisions in a 24-h period, leading to apoptosis of infected cells. Induction of apoptosis required bacterial internalization and is associated with intracellular replication. The slow and gradual replication of S. aureus inside epithelial cells hints at the role of host factors or signals in bacterial growth and further suggests possible cross talk between host cells and S. aureus.

Apoptosis↗