Biomedical subjects

K L Chua

Publications and source records attributed to K L Chua.

At least 19 recordsLinked to original sources

PubMed2004

BpeAB-OprB, a multidrug efflux pump in Burkholderia pseudomallei.

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to a wide range of antimicrobial agents, including beta-lactams, aminoglycosides, macrolides, and polymyxins. An operon, bpeR-bpeA-bpeB-oprB, which encodes a putative repressor, a membrane fusion protein, an inner membrane protein, and an outer membrane protein, respectively, of a multidrug efflux pump of the resistance-nodulation-division family was identified in B. pseudomallei. The divergently transcribed bpeR gene encodes a putative repressor protein of the TetR family which probably regulates the expression of the bpeAB-oprB gene cluster. Comparison of the MICs and minimal bactericidal concentrations of antimicrobials for bpeAB deletion mutant KHW Delta bpeAB and its isogenic wild-type parent, KHW, showed that the B. pseudomallei BpeAB-OprB pump is responsible for the efflux of the aminoglycosides gentamicin and streptomycin, the macrolide erythromycin, and the dye acriflavine. Antibiotic efflux by the BpeAB-OprB pump was dependent on a proton gradient and differs from that by the AmrAB-OprA pump in that it did not efflux the aminoglycoside spectinomycin or the macrolide clarithromycin. The broad-spectrum efflux pump inhibitor MC-207,110 did not potentiate the effectiveness of the antimicrobials erythromycin and streptomycin in B. pseudomallei.

Aminoglycosides↗

PubMed2003

Flagella are virulence determinants of Burkholderia pseudomallei.

Burkholderia pseudomallei, a facultatively intracellular pathogen, is a flagellated and motile gram-negative bacterium and is the causative agent of melioidosis in humans. Flagella are commonly recognized as important virulence determinants expressed by bacterial pathogens since the motility phenotype imparted by these organelles often correlates with the ability of an organism to cause disease. We used a virulent isolate of B. pseudomallei, KHW, to construct an isogenic deletion mutant with a mutation in the flagellin gene (fliC) by gene replacement transposon mutagenesis. The KHWDeltafliCKm mutant was aflagellate and nonmotile in semisolid agar. The isogenic KHWDeltafliCKm mutant was not impaired in terms of the ability to invade and replicate in cultured human lung cells compared with the wild type. It was also equally virulent in slow-killing assays involving Caenorhabditis elegans, but it was avirulent during intranasal infection of BALB/c mice. Very few bacteria, if any, were isolated from the lungs and spleens of KHWDeltafliCKm-infected mice. In contrast, the bacterial loads in the lungs and spleens were similar in mice infected with KHW and in mice infected with the complemented mutant, KHWDeltafliCKm/pUCP28TfliC. Unlike the Syrian hamster or diabetic rat models of infection, the B. pseudomallei flagellin was also a virulence factor during intraperitoneal infection of BALB/c mice. In this study, all animals infected with KHWDeltafliCKm remained healthy and did not succumb to disease regardless of the route of infection. The flagellum is therefore an important and necessary virulence determinant of B. pseudomallei during intranasal and intraperitoneal infection of mice.

Animals↗

PubMed2000

A new cytolysin from the sea anemone, Heteractis magnifica: isolation, cDNA cloning and functional expression.

We purified a new cytolysin (HMgIII) from the sea anemone, Heteractis magnifica. HMgIII, which has a molecular mass of approximately 19 kDa, functions as both a cytolysin and a hemolysin. The full-length HMg III cDNA was obtained by reverse transcriptase-polymerase chain reaction, using primers designed from its N-terminal amino acid sequence and an internal conserved region of two other sea anemone cytolysins: equinatoxin II (EqT II) and cytolysin III. The cDNA contained an open reading frame of 633 bp, which encodes a protein of 211 amino acids. The nascent HMg III protein contained a prepropeptide of 34 amino acids, which includes a signal peptide of 19 amino acids. The mature HMg III has a predicted molecular mass of 19 kDa and a pI of 9.1, and shares 91%, 89%, 65% and 63% amino acid sequence similarity with cytolysin III, cytolysin ST I, tenebrosin-C and equinatoxin (EqT II), respectively. The predicted secondary structure of the mature HMg III comprises 16% alpha-helix, 23% extended strand and 60% random coils. The characteristic amphiphilic alpha-helix of cytolysins is located at the N-terminus of the processed HMg III. Recombinant HMg III (rHMg III) was expressed in Escherichia coli as a fusion protein containing a 6xHisTag at the N-terminus. The hemolytic and cytotoxic activities of the purified rHMg III were comparable to those of the native HMg III. The hemolytic activities of both proteins were similarly potentiated with 8-anilino-1-naphthalenesulfonate (ANS). Increasing the length of the peptide tag on the N-terminal of rHMg III correlated with decreasing hemolytic activity, thus confirming the importance of the N-terminal amphiphilic alpha-helix for its cytolytic activity.

Amino Acid Sequence↗

PubMed2000

Type I and type II cytokeratin cDNAs from the zebrafish (Danio rerio) and expression patterns during early development.

Full-length cDNAs of a type I (zfCKI), and a type II (zfCKII) cytokeratin from the adult zebrafish, Danio rerio, were characterized and their expressions studied during early development and in the adult. The 1,426 bp long zfCKI cDNA encodes a 46.7 kD protein, whereas the 2,398 bp zfCKII cDNA encodes a protein of 58.6 kD. zfCKI and zfCKII each have a central rod domain that is characteristic of intermediate filaments and which share 73%-91% and 87%-93% similarity, respectively, with those of type I and type II cytokeratins from zebrafish, goldfish, and the rainbow trout. The central rod domains of zfCKI and zfCKII also contain the IF signature motif, IA[T/E]YR[K/R]LL[D/E]. zfCKI has, in addition, a leucine-zipper motif at a.a. residues 184-205 and 191-212. Both zfCKI and zfCKII mRNAs are expressed in the epidermis of the zebrafish. zfCKII mRNA was both maternally inherited and zygotically transcribed and was detected from the one-cell embryo to adult stages. zfCKII was also strongly expressed specifically during the 20-somites, protruding-mouth, and adult stages. In the adult, it was uniformly expressed in the skin, fins and scale epidermis. In contrast, zfCKI mRNA was undetectable in the oocyte but was zygotically transcribed from the epiboly stage onwards. Its expression in the skin was strong only up to the swimming larva stage and was weak and patchy in the adult. Both zfCKI and zfCKII were expressed in the neurons and glial cells of the brain and spinal cord. In the adult eye, zfCKI and zfCKII were expressed in the ganglion cell layer and the retina, but zfCKII was also strongly expressed in the cornea as well as in chondrocytes in the skull.

Aging↗

PubMed2000

Characterization of a zebrafish (Danio rerio) desmin cDNA: an early molecular marker of myogenesis.

Desmin is a muscle-specific protein and a constitutive subunit of the intermediate filaments (IF) in skeletal, cardiac and smooth muscles. It is an early marker of skeletal muscle myogenesis. We have characterized a clone of desmin cDNA from an embryonic zebrafish (Danio rerio) cDNA library. The full-length cDNA comprised 1798 nucleotides, encoding a protein of 473 amino acids. The predicted amino acid sequence of the zebrafish desmin shares a high degree of similarity to other vertebrate desmins, but also contains a sequence at the carboxyl terminal of the tail domain that is unique to the zebrafish. It carries many features which are distinctive of IF subunit proteins. These include the T/SSYRRXF/Y motif in the head domain, and the intermediate filament signature consensus, [I/V]-X-[T/A/C/I]-Y-[R/K/H]-X-[L/M]-L-[D/E], located in the carboxyl terminus of the central helical rod. Unlike other 3' UTR sequences, the 3' UTR of the zebrafish cDNA sequence has two CAYUG elements flanking a single polyadenylation site. The temporal and spatial expression patterns of desmin mRNA during early zebrafish development were studied. The onset of desmin expression occurred at the 1-3 somite stage (11 hpf). It increased throughout somitogenesis, with maximum expression at the Prim-6 stage (25 hpf), and decreasing expression towards the protruding-mouth stage (72 hpf). Desmin mRNA was initially localised exclusively to the somites, but was subsequently also detected in other musculature in the developing heart and fins. The onset of expression and the spatial localization of desmin mRNA in the zebrafish coincides with that reported for MyoD and myogenin.

Amino Acid Sequence↗

PubMed2000

Characterization of a predominant immunogenic outer membrane protein of Riemerella anatipestifer.

The ompA gene, encoding the 42-kDa major antigenic outer membrane protein OmpA of Riemerella anatipestifer, the etiololgical agent of septicemia anserum exsudativa, was cloned and expressed in Escherichia coli. Recombinant OmpA displayed a molecular mass similar to that predicted from the nucleotide sequence of the ompA gene but lower than that observed in total cell lysates of R. anatipestifer. The ompA gene showed a conserved C-terminal region comprising the OmpA-like domain and a variable N-terminal region. This structure is similar to those of the analogous outer membrane proteins of several gram-negative bacteria. However, OmpA of R. anatipestifer contains six EF-hand calcium-binding domains and two PEST regions, which distinguish it from other outer membrane proteins. The occurrence of these motifs in OmpA suggests a possible role in virulence for this protein. The ompA gene is present in the R. anatipestifer type strain and in all serotype reference strains. However, it exhibits some minor genetic heterogeneity among different serotypes, which seems not to affect the strong antigenic characteristics of the protein. OmpA is a conserved and strong antigenic determinant of R. anatipestifer and hence is suggested to be a valuable protein for the serodetection of R. anatipestifer infections, independent of their serotype.

Amino Acid Sequence↗

PubMed1999

Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR.

Riemerella anatipestifer is a gram-negative rod-shaped bacterium associated with epizootic infections in poultry. A total of 35 R. anatipestifer isolates including the type strain ATCC11845T, reference and field strains for 18 different serotypes were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence. This technique was applied by using either extracted genomic DNA or preparation of whole bacterial cells harvested directly from plate cultures. Rep-PCR discriminated the R. anatipestifer isolates into 19 electrophoretic types. DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns. Substantial variation was seen among the rep-PCR fingerprints of different serotypes. Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype. These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R. anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype. Rep-PCR may serve as a useful molecular tool for subtyping R. anatipestifer isolates for epidemiologic investigations. The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells.

Animals↗

PubMed1997

Human papillomavirus analysis as a prognostic marker following conization of the cervix uteri.

Cervical intraepithelial neoplasia grade 3 (CIN 3) is treated surgically. Follow-up of these patients is important to ensure successful treatment. The present study was undertaken to determine whether human papillomavirus (HPV) testing can be used to discriminate patients who will have recurrences from those who will not. It is composed of 26 patients who presented with recurrences of CIN and 22 patients who remained disease-free after treatment. DNA was extracted from paraffin-embedded cone biopsies of incident CIN 3, their corresponding follow-up Pap smears taken 3 months postoperatively, and their secondary cone biopsies of the recurrent lesions. The extracted DNA were then analyzed by PCR for the presence of HPV. The posttreatment cervical smears in the recurrent group had a (25/26) 96% HPV prevalence, while HPV DNA was not detectable in any of the 22 patients in the control group. The HPV types in both the initial and recurrent lesions correlated very well. This suggest that most recurrences are likely to be due to persisting lesions or subclinical HPV infections that had not been completely removed. Cytology alone was not sufficiently sensitive to discriminate the patients at risk for recurrences. It appears that HPV testing can be useful to monitor the therapeutic result.

Adult↗

PubMed1997

Phylogenetic position of Riemerella anatipestifer based on 16S rRNA gene sequences.

Riemerella anatipestifer, the causative agent of septicemia anserum exsudativa (also called new duckling disease), belongs to the family Flavobacteriaceae of gram-negative bacteria. We determined the DNA sequences of the rrs genes encoding the 16S rRNAs of four R. anatipestifer strains by directly sequencing PCR-amplified rrs genes. A sequence similarity analysis confirmed the phylogenetic position of R. anatipestifer in the family Flavobacteriaceae in rRNA superfamily V and allowed fine mapping of R. anatipestifer on a separate rRNA branch comprising the most closely related species, Bergeyella zoohelcum, as well as Chryseobacterium balustinum, Chryseobacterium indologenes, and Chryseobacterium gleum. The sequences of the rrs genes of the four R. anatipestifer strains varied between 0.5 and 1.0%, but all of the strains occupied the same position on the phylogenetic tree. In general, differences in rrs genes were observed among R. anatipestifer strains, even within a given serotype, as shown by restriction fragment length polymorphism of PCR-amplified rrs genes.

Animals↗

PubMed1996

A prospective study on the risk of cervical intra-epithelial neoplasia among healthy subjects with serum antibodies to HPV compared with HPV DNA in cervical smears.

To estimate the risk of developing cervical intra-epithelial neoplasia (CIN) among women exposed to human papillomavirus (HPV) type 16, we performed a prospective study in a population-based cohort of more than 15,000 women followed for 34.9 months. Seventy-four women developed CIN during follow-up and were matched for age, time of sampling and area of residence with 148 women who remained CIN-free during follow-up. The blood samples taken at enrollment were tested for serum antibodies to HPV types 16, 18 and 33 capsids. Cervical smears or biopsies were analyzed for the presence of HPV DNA by nested PCR using HPV general primers and by HPV 16- and 18-type-specific PCR HPV serology and HPV-PCR were in good agreement, particularly when the blood sample and the Pap smear were taken less than 6 months apart. HPV DNA was found in 88% of cases and 4% of controls, whereas HPV 16 DNA was present in 44% of cases and in 1 of 142 controls. HPV-16-seropositive women had a 3-fold increased risk of developing CIN. The risk was highest among women younger than 35 years of age, of whom an estimated 3.4% of HPV-16-seropositive and 0.5% of seronegative women developed CIN. Since the risk associated with HPV-16 seropositivity (a measure of past or present infection) was 35-fold lower than that of HPV DNA (present infection), most infections appear to be eliminated before CIN develops. In conclusion, HPV 16 infection does confer an excess risk of CIN development, and HPV DNA detection has a high predictive value for the presence of high-grade CIN.

Adult↗

PubMed1996

Persistence of human papillomavirus (HPV) infections preceding cervical carcinoma.

BACKGROUND: A persistent genital infection with an oncogene-type of human papillomavirus (HPV) is considered to be essential for the development of most cervical carcinomas. Therefore, HPV analysis has been proposed as a possible complementary cytological screening program. The authors have developed a technique to analyze archival Pap smears, which has enabled them to study the relation between persistent HPV infection and the development of cervical cancer. METHODS: Nested polymerase chain reaction was used to demonstrate the presence of HPV DNA, and sequencing of the obtained amplimer was performed to establish HPV type. The authors analyzed a series of 88 smears taken 1.5 to 7 years prior to the diagnosis of an HPV-containing cervical carcinoma (12 invasive adenocarcinomas, 18 invasive squamous carcinomas, and 58 squamous carcinoma in situ), which were compared with age-matched controls with no tumor development. RESULTS: HPV DNA was present in a majority of the smears preceding a cancer, with an odds ratio of around 15 for all tumor groups. Infections with a persisting HPV type were demonstrated in most samples from a series of eight patients, from who multiple smears were available, covering the 7-year period preceding the cancer diagnosis. CONCLUSIONS: Persisting infection can be demonstrated in exfoliated cells many years before cancer is diagnosed. The results are complementary to those obtained with cytology, that is, HPV is detected also in those at-risk patients whose Pap smears are morphologically normal. However, the results are still insufficient to justify a general recommendation to use HPV testing for health control purposes.

Adenocarcinoma↗

PubMed1996

Cytological evaluation and molecular human papillomavirus test of cervical scrapings from women treated for condyloma.

The incidence of cervical carcinoma has decreased by about half since cytological screening was introduced in Sweden in the 1960s. This is an encouraging but not altogether satisfactory development. Human papillomaviruses (HPVs) are known to be potential agents in the etiology of cervical cancer. Therefore, an additional HPV test might well improve the detection rate of high-grade cervical intraepithelial neoplasia (CIN). The purpose of this investigation was to compare the correlation of cytology and HPV testing in a pilot study of 94 women recruited from a clinic for sexually transmitted diseases after condyloma treatment and to check earlier established molecular biological assays. Cervical scrapings, taken for simultaneous Pap smear and molecular HPV DNA testing, were assessed by the polymerase chain reaction. Of the 94 women tested, 47 (50%) had normal cytology and negative HPV DNA; 36 (38%) had normal cytology but positive HPV DNA, 26 (72%) of whom harbored high-risk HPVs; 1 (1%) had abnormal cytology but negative HPV DNA, and 10 (11%) had abnormal cytology and positive HPV DNA, 5 (50%) of whom harbored high-risk HPVs. It is concluded that an HPV test would add greater specificity and possibly also greater sensitivity to cytology for detecting or predicting high-grade CIN. This information may be of value when designing future gynecological screening programs.

Adult↗

PubMed1996

A clinical and laboratory evaluation of three types of operating gloves for use in orthodontic practice.

This prospective study examined the durability and user acceptability of three types of operating glove for use in orthodontic practice. The glove types studied were a lightweight examination glove (Microtouch, Johnson and Johnson), a dedicated dental procedure glove (Biogel D, Regent Hospital Products) and a non-latex, nitrile glove (N-Dex, Best Manufacturing Europe N.V.). A water-leak test was used to examine 50 pairs of unused gloves of each type for manufacturing defects. Subsequently, six operators of varying experience were asked to use 20 pairs of each type of glove for fixed appliance treatment sessions and record their suitability for different clinical procedures. The used gloves were collected and tested in the laboratory for punctures. At the end of the study the six operators completed a detailed questionnaire recording their preferences for each glove type.

Attitude of Health Personnel↗

PubMed1995

Polymerase chain reaction analysis of human papillomavirus in archival cervical cytologic smears.

We used a nested polymerase chain reaction (PCR) to study the presence of human papillomavirus (HPV) DNA in stained, archival cervical cytologic smears, where MY09/MY11 served as outer primers and GP5/GP6 as inner primers. It was found to give a higher positivity rate than PCR using the E1 degenerate consensus primers, where the sensitivity was decreased to 80%. It seemed optimal to use less sample DNA (0.5%) for the reaction; larger volumes resulted in decreased reactivity. Similarly, the presence of bovine serum albumin helped to improve the reactivity. The risk of cross-contamination did not seem to be a major obstacle to a valid analysis. The prevalence of HPV in normal smears was 10%, and in the high grade squamous intraepithelial lesion group it was 80%. Smears with cytologic evidence of HPV gave 100% positivity, while those containing cancer cells gave 80%. In patients whose Southern blot had demonstrated the presence of HPV, 87% of the simultaneously taken smears were also positive with nested PCR. Similarly, in those whose Southern blot analysis was negative, the corresponding smear was positive in 41%; this reactivity was associated with simultaneous squamous intraepithelial lesions. The prevalence values indicated that this analysis is both sensitive and specific and that it can be used to evaluate the performance of other diagnostic methods. The validity is sufficient to allow retrospective cohort studies of the natural history of HPV infections during carcinogenesis.

Blotting, Southern↗

PubMed1994

Complete androgen insensitivity due to a splice-site mutation in the androgen receptor gene and genetic screening with single-stranded conformation polymorphism.

OBJECTIVE: To characterize the genetic defect in a family with complete androgen insensitivity syndrome and to determine whether single-stranded conformation polymorphism (SSCP) can be used to detect subtle mutations in the androgen receptor (AR) gene. DESIGN: Amplification, subcloning where appropriate, and sequencing of the AR gene in members of the affected family and to use SSCP to differentiate rapidly mutant from normal alleles. SETTING: Reproductive endocrinology clinic and laboratory in a university hospital. PATIENTS: A family of which two sisters (46XY) have complete androgen insensitivity syndrome. RESULTS: A novel single base (G --> A) mutation in the exon G-intron 7 junction of the AR gene caused an abnormal donor splice site leading to complete androgen insensitivity in both affected siblings. Their mother was demonstrated to be the heterozygous carrier of this mutation while the other two males in the family carried the normal allele. Single-stranded conformation polymorphism proved useful for defining the normal, mutant, and heterozygous carrier status of each member of this family. CONCLUSIONS: This new mutation of the human AR gene illustrates the importance of exon G in receptor function. Single-stranded conformation polymorphism is a simple and rapid screening technique that can be used to detect unknown subtle mutations in the AR gene.

Adolescent↗

PubMed1994

Detection of the BC 24 transforming fragment of the herpes simplex virus type 2 (HSV-2) DNA in cervical carcinoma tissue by polymerase chain reaction (PCR).

The polymerase chain reaction (PCR) was used to investigate samples from Indonesian and Swedish patients with cervical intraepithelial neoplasia grade III (CIN III), squamous cell carcinoma or adenocarcinoma of the cervix for the presence of a transforming fragment (BC 24) of herpes simplex virus type 2 (HSV-2) DNA. The PCR test for HSV-2 DNA was more sensitive than the infectivity endpoint titer in a cell culture system and no cross reactivity was found with either varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, human papillomavirus 16 or 18, or human genomic DNA. Using this PCR test, 2 out of 5 cases with CIN III, 10 of 71 squamous cell carcinomas, and 3 of 11 adenocarcinomas of the uterine cervix were found to contain DNA sequences homologous to the BC 24 fragment of the HSV-2 genome. Only two of the samples containing this transforming region of the HSV-2 DNA were positive in a PCR assay for the HSV-2 DNA polymerase gene. The great majority of the HSV-2 BC 24 DNA positive (12 of 15) came from the Indonesian group of patients. All 15 CIN III or cancer samples positive for the HSV-2 BC 24 fragment were also positive for papillomavirus DNA. In line with observations made by others, our data support the hypothesis that HSV infection could represent one of several possible oncogenic cofactors leading to cervical carcinoma. The HSV cofactor might be more important in the Indonesian than in the Swedish population.

Adenocarcinoma↗

PubMed1994

Echocardiographic detection of mobile right atrial thrombus.

Mobile right atrial thrombus is a rare echocardiographic diagnosis with important implications on further management. We report a case of 50-year-old woman with deep vein thrombosis of her left lower limb, who was found to have a large mobile serpiginous right atrial thrombus on both two-dimensional echocardiography (2DE) and transoesophageal echocardiography (TEE). She developed acute pulmonary embolism within thirty-six hours of diagnosis and subsequently underwent successful pulmonary embolectomy. This case demonstrates the usefulness of echocardiography as a non-invasive tool in the investigation of suspected pulmonary thromboembolism. The detection of right atrial thrombus mandates serious consideration for prompt surgical action.

Echocardiography↗

PubMed1993

Expression of the recA gene in recombination-deficient (rec-) strains of Escherichia coli.

Basal and induced levels of recA expression in wild-type and isogenic derivatives of Escherichia coli carrying various rec mutations were measured using a low-copy number recApo-lacZ fusion, pKLC3.2. Basal recA expression in wild-type and isogenic derivatives containing single rec- mutations, as well as in the recBCsbcA strain and isogenic recA, recF and recJ derivatives, ranged from 1000 to 3900 units. In the recBCsbcBC strain and isogenic recL and recN derivatives basal recA expressions were 3- to 5-fold higher than that of wild-type cells and were inducible by mitomycin C. Except for the recA and lexA3(lnd-) mutants, recA expression was induced by mitomycin C in wild-type cells and its isogenic recB, recD, recF, recG, recJ, recL, recN, recO and ruv derivatives. RecF was required for induction of recA expression by mitomycin C, but not by naladixic acid in the recBCsbcA and recBCsbcBC genetic backgrounds. In wild-type cells, induction of recA expression by naladixic acid required the recBC, but not the recD function of the RecBCD enzyme. This requirement is suppressed by either an additional sbcA or sbcC mutation, but not by an sbcB mutation.

Cloning, Molecular↗