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  1. Rapamycin synergizes cisplatin sensitivity in basal-like breast cancer cells through up-regulation of p73
    Wong SW, Tiong KH, Kong WY, Yue YC, Chua CH, Lim JY, et al.
    Breast Cancer Res Treat, 2011 Jul;128(2):301-13.
    PMID: 20686837 DOI: 10.1007/s10549-010-1055-0
    Recent gene expression profiling studies have identified five breast cancer subtypes, of which the basal-like subtype is the most aggressive. Basal-like breast cancer poses serious clinical challenges as there are currently no targeted therapies available to treat it. Although there is increasing evidence that these tumors possess specific sensitivity to cisplatin, its success is often compromised due to its dose-limiting nephrotoxicity and the development of drug resistance. To overcome this limitation, our goal was to maximize the benefits associated with cisplatin therapy through drug combination strategies. Using a validated kinase inhibitor library, we showed that inhibition of the mTOR, TGFβRI, NFκB, PI3K/AKT, and MAPK pathways sensitized basal-like MDA-MB-468 cells to cisplatin treatment. Further analysis demonstrated that the combination of the mTOR inhibitor rapamycin and cisplatin generated significant drug synergism in basal-like MDA-MB-468, MDA-MB-231, and HCC1937 cells but not in luminal-like T47D or MCF-7 cells. We further showed that the synergistic effect of rapamycin plus cisplatin on basal-like breast cancer cells was mediated through the induction of p73. Depletion of endogenous p73 in basal-like cells abolished these synergistic effects. In conclusion, combination therapy with mTOR inhibitors and cisplatin may be a useful therapeutic strategy in the treatment of basal-like breast cancers.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  2. A B-myb--DREAM complex is not critical to regulate the G2/M genes in HPV-transformed cell lines
    Rashid NN, Yusof R, Watson RJ
    Anticancer Res, 2014 Nov;34(11):6557-63.
    PMID: 25368258
    It is well-established that HPV E7 proteins, encoded by human papillomavirus (HPV) genes, frequently associated with cervical cancers bind avidly to the retinoblastoma (RB) family of pocket proteins and disrupt their association with members of the E2F transcription factor family. Our previous study showed that the repressive p130-dimerization partner, RB-like, E2F and multi-vulval class (DREAM) complex was disrupted by HPV16 E7 proteins in order to maintain the viral replication in CaSki cells. However, we would like to address whether the activator B-myb-DREAM complex is critical in regulating the replication and mitosis phase since our previous study showed increased B-myb-DREAM expression in HPV-transformed cell lines when compared to control cells.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  3. Sexual maturation modulates expression of nuclear receptor types in laser-captured single cells of the cichlid (Oreochromis niloticus) pituitary
    Kitahashi T, Ogawa S, Soga T, Sakuma Y, Parhar I
    Endocrinology, 2007 Dec;148(12):5822-30.
    PMID: 17823257
    The role of steroid/thyroid hormones in the regulation of endocrine cells at the level of the pituitary has remained unclear. Therefore, using single-cell quantitative real-time PCR, we examined absolute amounts of transcripts for nuclear receptors [estrogen receptors (ERs) alpha, beta, and gamma; androgen receptors (ARs) a and b; glucocorticoid receptors (GRs) 1, 2a, and 2b; and thyroid hormone receptors (TRs) alpha1, alpha2, and beta] in pituitary cells of immature (IM) and mature (M) male tilapia, Oreochromis niloticus. In the two reproductive stages, ACTH cells expressed only ERbeta, whereas all other pituitary cell types expressed ERalpha + beta, and a subpopulation coexpressed ARa, ARb, GR1, GR2b, and TRbeta but lacked ERgamma, GR2a, TRalpha1, and TRalpha2. IM males had high percentages of LH cells (IM 46.0% vs. M 10.0%), GH cells (IM 23.3% vs. M 7.9%), and prolactin cells (IM 68.8% vs. M 6.0%) with ERbeta, and TSH cells (IM 19.2% vs. M 0.0%) and MSH cells (IM 25.6% vs. M 0.0%) with ERalpha + TRbeta. A high percentage of FSH cells in IM males expressed ERbeta (IM 46.9% vs. M 18.8%), and FSH cells in M males showed significantly high GR1 transcripts (IM 76.0 +/- 5.0 vs. M 195.0 +/- 10.7 copies per cell; P < 0.05), suggesting that FSH cells are regulated differently in the two reproductive stages. Coexpression of ERalpha + beta in high percentages of cells of the GH family (GH, IM 43.8% vs. M 14.3%; prolactin, IM 8.3% vs. M 59.7%; somatolactin, IM 22.2% vs. M 42.2%) suggests that the expression of both ERs is important for functionality. Thus, differential coexpression of genes for nuclear receptors in subpopulations of pituitary cell types suggests multiple steroid/thyroid hormone regulatory pathways at the level of the pituitary during the two reproductive stages.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  4. Generating neuron-like cells from BM-derived mesenchymal stromal cells in vitro
    Choong PF, Mok PL, Cheong SK, Leong CF, Then KY
    Cytotherapy, 2007;9(2):170-83.
    PMID: 17453969
    The multipotency of stromal cells has been studied extensively. It has been reported that mesenchymal stromal cells (MSC) are capable of differentiating into cells of multilineage. Different methods and reagents have been used to induce the differentiation of MSC. We investigated the efficacy of different growth factors in inducing MSC differentiation into neurons.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  5. Chemical composition and cytotoxic properties of Clinacanthus nutans root extracts
    Teoh PL, Cheng AY, Liau M, Lem FF, Kaling GP, Chua FN, et al.
    Pharm Biol, 2017 Dec;55(1):394-401.
    PMID: 27931178
    CONTEXT: Clinacanthus nutans Lindau (Acanthaceae) is a medicinal plant that has been reported to have anti-inflammatory, antiviral, antimicrobial and antivenom activities. In Malaysia, it has been widely claimed to be effective in various cancer treatments but scientific evidence is lacking.

    OBJECTIVE: This study investigates the chemical constituents, anti-proliferative, and apoptotic properties of C. nutans root extracts.

    MATERIALS AND METHODS: The roots were subjected to solvent extraction using methanol and ethyl acetate. The anti-proliferative effects of root extracts were tested at the concentrations of 10 to 50 μg/mL on MCF-7 and HeLa by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for 72 h. Morphological changes were observed under light microscope. Pro-apoptotic effects of root extracts were examined using flow cytometric analysis and RT-PCR. The chemical compositions of root extracts were detected using GC-MS.

    RESULTS: The proliferation of MCF-7 cells was inhibited with the IC50 values of 35 and 30 μg/mL, respectively, for methanol and ethyl acetate root extracts. The average inhibition of HeLa cells was ∼25%. Induction of apoptosis in MCF-7 was supported by chromatin condensation, down-regulation of BCL2 and unaltered expression of BAX. However, only ethyl acetate extract caused the loss of mitochondrial membrane potential. GC-MS analysis revealed the roots extracts were rich with terpenoids and phytosterols.

    DISCUSSION AND CONCLUSIONS: The results demonstrated that root extracts promote apoptosis by suppressing BCL2 via mitochondria-dependent or independent manner. The identified compounds might work solely or cooperatively in regulating apoptosis. However, further studies are required to address this.

    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  6. Fulltext Component-specific effectiveness of trivalent influenza vaccine as monitored through a sentinel surveillance network in Canada, 2006-2007
    Skowronski DM, De Serres G, Dickinson J, Petric M, Mak A, Fonseca K, et al.
    J Infect Dis, 2009 Jan 15;199(2):168-79.
    PMID: 19086914 DOI: 10.1086/595862
    Trivalent inactivated influenza vaccine (TIV) is reformulated annually to contain representative strains of 2 influenza A subtypes (H1N1 and H3N2) and 1 B lineage (Yamagata or Victoria). We describe a sentinel surveillance approach to link influenza variant detection with component-specific vaccine effectiveness (VE) estimation.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  7. Cortisol affects metabolic and ionoregulatory responses to a different extent depending on feeding ration in common carp, Cyprinus carpio
    Liew HJ, Fazio A, Faggio C, Blust R, De Boeck G
    Comp Biochem Physiol A Mol Integr Physiol, 2015 Nov;189:45-57.
    PMID: 26219478 DOI: 10.1016/j.cbpa.2015.07.011
    Interacting effects of feeding and stress on corticoid responses in fish were investigated in common carp fed 3.0% or 0.5% body mass (BM) which received no implant, a sham or a cortisol implant (250 mg/kg BM) throughout a 168 hour post-implant period (168 h-PI). At 12h-PI, cortisol implants elevated plasma cortisol, glucose and lactate. Plasma osmolality and ions remained stable, but cortisol increased gill and kidney Na(+)/K(+) ATPase (NKA) and H(+) ATPase activities. Gill NKA activities were higher at 3%-BM, whereas kidney H(+) ATPase activity was greater at 0.5%-BM. Cortisol induced liver protein mobilization and repartitioned liver and muscle glycogen. At 3%-BM, this did not increase plasma ammonia, reflecting improved excretion efficiency concomitant with upregulation of Rhesus glycoprotein Rhcg-1 in gill. Responses in glucocorticoid receptors (GR1/GR2) and mineralocorticoid receptor (MR) to cortisol elevation were most prominent in kidney with increased expression of all receptors at 24 h-PI at 0.5%-BM, but only GR2 and MR at 0.5%-BM. In the liver, upregulation of all receptors occurred at 24 h-PI at 3%-BM, whilst only GR2 and MR were upregulated at 0.5%-BM. In the gill, there was a limited upregulation: GR2 and MR at 72 h-PI and GR1 at 168 h-PI at 3%-BM but only GR2 at 72 h-PI at 0.5%-BM. Thus cortisol elevation led to similar expression patterns of cortisol receptors in both feeding regimes, while feeding affected the type of receptor that was induced. Induction of corticoid receptors occurred simultaneously with increases in Rhcg-1 mRNA expression (gill) but well after NKA and H(+) ATPase activities increased (gill/kidney).
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  8. Fulltext Soluble factors from stellate cells induce pancreatic cancer cell proliferation via Nrf2-activated metabolic reprogramming and ROS detoxification
    Wu YS, Looi CY, Subramaniam KS, Masamune A, Chung I
    Oncotarget, 2016 Jun 14;7(24):36719-36732.
    PMID: 27167341 DOI: 10.18632/oncotarget.9165
    Pancreatic stellate cells (PSC), a prominent stromal cell, contribute to the progression of pancreatic ductal adenocarcinoma (PDAC). We aim to investigate the mechanisms by which PSC promote cell proliferation in PDAC cell lines, BxPC-3 and AsPC-1. PSC-conditioned media (PSC-CM) induced proliferation of these cells in a dose- and time-dependent manner. Nrf2 protein was upregulated and subsequently, its transcriptional activity was increased with greater DNA binding activity and transcription of target genes. Downregulation of Nrf2 led to suppression of PSC-CM activity in BxPC-3, but not in AsPC-1 cells. However, overexpression of Nrf2 alone resulted in increased cell proliferation in both cell lines, and treatment with PSC-CM further enhanced this effect. Activation of Nrf2 pathway resulted in upregulation of metabolic genes involved in pentose phosphate pathway, glutaminolysis and glutathione biosynthesis. Downregulation and inhibition of glucose-6-phosphate-dehydrogenase with siRNA and chemical approaches reduced PSC-mediated cell proliferation. Among the cytokines present in PSC-CM, stromal-derived factor-1 alpha (SDF-1α) and interleukin-6 (IL-6) activated Nrf2 pathway to induce cell proliferation in both cells, as shown with neutralization antibodies, recombinant proteins and signaling inhibitors. Taken together, SDF-1α and IL-6 secreted from PSC induced PDAC cell proliferation via Nrf2-activated metabolic reprogramming and ROS detoxification.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  9. Fulltext Ankrd11 is a chromatin regulator involved in autism that is essential for neural development
    Gallagher D, Voronova A, Zander MA, Cancino GI, Bramall A, Krause MP, et al.
    Dev. Cell, 2015 Jan 12;32(1):31-42.
    PMID: 25556659 DOI: 10.1016/j.devcel.2014.11.031
    Ankrd11 is a potential chromatin regulator implicated in neural development and autism spectrum disorder (ASD) with no known function in the brain. Here, we show that knockdown of Ankrd11 in developing murine or human cortical neural precursors caused decreased proliferation, reduced neurogenesis, and aberrant neuronal positioning. Similar cellular phenotypes and aberrant ASD-like behaviors were observed in Yoda mice carrying a point mutation in the Ankrd11 HDAC-binding domain. Consistent with a role for Ankrd11 in histone acetylation, Ankrd11 was associated with chromatin and colocalized with HDAC3, and expression and histone acetylation of Ankrd11 target genes were altered in Yoda neural precursors. Moreover, the Ankrd11 knockdown-mediated decrease in precursor proliferation was rescued by inhibiting histone acetyltransferase activity or expressing HDAC3. Thus, Ankrd11 is a crucial chromatin regulator that controls histone acetylation and gene expression during neural development, thereby providing a likely explanation for its association with cognitive dysfunction and ASD.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  10. Blood gene signature for early hepatocellular carcinoma detection in patients with chronic hepatitis B
    Omar H, Lim CR, Chao S, Lee MM, Bong CW, Ooi EJ, et al.
    J Clin Gastroenterol, 2015 Feb;49(2):150-7.
    PMID: 25569223 DOI: 10.1097/MCG.0000000000000112
    Up to 25% of chronic hepatitis B (CHB) patients eventually develop hepatocellular carcinoma (HCC), a disease with poor prognosis unless detected early. This study identifies a blood-based RNA biomarker panel for early HCC detection in CHB.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  11. Fulltext Testosterone reduces knee passive range of motion and expression of relaxin receptor isoforms via 5α-dihydrotestosterone and androgen receptor binding
    Dehghan F, Muniandy S, Yusof A, Salleh N
    Int J Mol Sci, 2014;15(3):4619-34.
    PMID: 24642882 DOI: 10.3390/ijms15034619
    Ovarian steroids such as estrogen and progesterone have been reported to influence knee laxity. The effect of testosterone, however, remains unknown. This study investigated the effect of testosterone on the knee range of motion (ROM) and the molecular mechanisms that might involve changes in the expression of relaxin receptor isoforms, Rxfp1 and Rxfp2 in the patella tendon and lateral collateral ligament of the female rat knee. Ovariectomized adult female Wistar rats received three days treatment with peanut oil (control), testosterone (125 and 250 μg/kg) and testosterone (125 and 250 μg/kg) plus flutamide, an androgen receptor blocker or finasteride, a 5α-reductase inhibitor. Duplicate groups received similar treatment however in the presence of relaxin (25 ng/kg). A day after the last drug injection, knee passive ROM was measured by using a digital miniature goniometer. Both tendon and ligament were harvested and then analysed for protein and mRNA expression for Rxfp1 and Rxfp2 respectively. Knee passive ROM, Rxfp1 and Rxfp2 expression were significantly reduced following treatment with testosterone. Flutamide or finasteride administration antagonized the testosterone effect. Concomitant administration of testosterone and relaxin did not result in a significant change in knee ROM as compared to testosterone only treatment; however this was significantly increased following flutamide or finasteride addition. Testosterone effect on knee passive ROM is likely mediated via dihydro-testosterone (DHT), and involves downregulation of Rxfp1 and Rxfp2 expression, which may provide the mechanism underlying testosterone-induced decrease in female knee laxity.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  12. Fulltext Molecular epidemiology of enterovirus 71 infection in the central region of Taiwan from 2002 to 2012
    Wu WH, Kuo TC, Lin YT, Huang SW, Liu HF, Wang J, et al.
    PLoS One, 2013;8(12):e83711.
    PMID: 24391812 DOI: 10.1371/journal.pone.0083711
    Enterovirus 71 (EV71), a causative agent of hand, foot, and mouth disease can be classified into three genotypes and many subtypes. The objectives of this study were to conduct a molecular epidemiological study of EV71 in the central region of Taiwan from 2002-2012 and to test the hypothesis that whether the alternative appearance of different EV71 subtypes in Taiwan is due to transmission from neighboring countries or from re-emergence of pre-existing local strains. We selected 174 EV71 isolates and used reverse transcription-polymerase chain reaction to amplify their VP1 region for DNA sequencing. Phylogenetic analyses were conducted using Neighbor-Joining, Maximum Likelihood and Bayesian methods. We found that the major subtypes of EV71 in Taiwan were B4 for 2002 epidemic, C4 for 2004-2005 epidemic, B5 for 2008-2009 epidemic, C4 for 2010 epidemic and B5 for 2011-2012 epidemic. Phylogenetic analysis demonstrated that the 2002 and 2008 epidemics were associated with EV71 from Malaysia and Singapore; while both 2010 and 2011-2012 epidemics originated from different regions of mainland China including Shanghai, Henan, Xiamen and Gong-Dong. Furthermore, minor strains have been identified in each epidemic and some of them were correlated with the subsequent outbreaks. Therefore, the EV71 infection in Taiwan may originate from pre-existing minor strains or from other regions in Asia including mainland China. In addition, 101 EV71 isolates were selected for the detection of new recombinant strains using the nucleotide sequences spanning the VP1-2A-2B region. No new recombinant strain was found. Analysis of clinical manifestations showed that patients infected with C4 had significantly higher rates of pharyngeal vesicles or ulcers than patients infected with B5. This is the first study demonstrating that different EV 71 genotypes may have different clinical manifestations and the association of EV71 infections between Taiwan and mainland China.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  13. Expression of rice thaumatin-like protein gene in transgenic banana plants enhances resistance to fusarium wilt
    Mahdavi F, Sariah M, Maziah M
    Appl Biochem Biotechnol, 2012 Feb;166(4):1008-19.
    PMID: 22183565 DOI: 10.1007/s12010-011-9489-3
    The possibility of controlling Fusarium wilt--caused by Fusarium oxysporum sp. cubensec (race 4)--was investigated by genetic engineering of banana plants for constitutive expression of rice thaumatin-like protein (tlp) gene. Transgene was introduced to cauliflower-like bodies' cluster, induced from meristemic parts of male inflorescences, using particle bombardment with plasmid carrying a rice tlp gene driving by the CaMV 35S promoter. Hygromycin B was used as the selection reagent. The presence and integration of rice tlp gene in genomic DNA confirmed by PCR and Southern blot analyses. RT-PCR revealed the expression of transgene in leaf and root tissues in transformants. Bioassay of transgenic banana plants challenged with Fusarium wilt pathogen showed that expression of TLP enhanced resistance to F. oxysporum sp. cubensec (race 4) compared to control plants.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  14. Genome-wide analysis of oral squamous cell carcinomas revealed over expression of ISG15, Nestin and WNT11
    Vincent-Chong VK, Ismail SM, Rahman ZA, Sharifah NA, Anwar A, Pradeep PJ, et al.
    Oral Dis, 2012 Jul;18(5):469-76.
    PMID: 22251088 DOI: 10.1111/j.1601-0825.2011.01894.x
    Multistep pathways and mechanisms are involved in the development of oral cancer. Chromosomal alterations are one of such key mechanisms implicated oral carcinogenesis. Therefore, this study aims to determine the genomic copy number alterations (CNAs) in oral squamous cell carcinoma (OSCC) using array comparative genomic hybridization (aCGH) and in addition attempt to correlate CNAs with modified gene expression.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  15. Isolation and characterization of differentially expressed transcripts from the suspension cells of oil palm (Elaeis guineensis Jacq.) in response to different concentration of auxins
    Roowi SH, Ho CL, Alwee SS, Abdullah MO, Napis S
    Mol Biotechnol, 2010 Sep;46(1):1-19.
    PMID: 20390382 DOI: 10.1007/s12033-010-9262-9
    Oil palm suspension cultures were initiated by transferring the gel-like friable embryogenic tissue onto liquid medium supplemented with auxins. In this study, transcripts that were differentially expressed in oil palm suspension cells cultured at different auxin concentrations were examined using suppression subtractive hybridization. Total RNA was first isolated from oil palm suspension cells proliferated in liquid medium with different hormone concentrations for 6 months. Four different hormone combinations: T1 (0.1 mg/l 2,4-D and 1.0 mg/l NAA), T2 (0.4 mg/l 2,4-D and 1.0 mg/l NAA), T3 (1.0 mg/l NAA), and T4 (0.4 mg/l 2,4-D) were used for the treatments. The first and second subtractions were performed using samples T1 and T2 in forward and reverse order. The other two subtractions were forward and reverse subtractions of T3 and T4, respectively. Reverse northern analyses showed that 14.13% of these clones were preferentially expressed in T1, 13.70% in T2, 14.75% in T3, and 15.70% in T4. Among the 294 cDNA clones that were sequenced, 61 contigs (assembled from 165 sequences) and 129 singletons were obtained. Among the 61 contigs, 10 contigs consist of sequences from treatment T1, 8 contigs were from treatment T2, 10 contigs were contains sequences of treatment T3 and 13 contigs contains sequences of treatment T4. Northern analyses of five transcripts that were shown to be differentially expressed in the oil palm suspension cells by reverse northern analysis revealed that transcripts 16A1 (a putative lignostilbene-alpha,beta-dioxygenase, EgLSD) and 16H12 (a putative ethylene responsive 6, EgER6) were differentially expressed in oil palm suspension cells treated with different levels of auxin.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  16. Regression of cervical intraepithelial neoplasia by zerumbone in female Balb/c mice prenatally exposed to diethylstilboestrol: involvement of mitochondria-regulated apoptosis
    Abdelwahab SI, Abdul AB, Devi N, Taha MM, Al-zubairi AS, Mohan S, et al.
    Exp. Toxicol. Pathol., 2010 Sep;62(5):461-9.
    PMID: 19581075 DOI: 10.1016/j.etp.2009.06.005
    Cervical cancer is the second most common cause of cancer death in women. We have demonstrated previously that zerumbone (ZER) has an anti-cancer effect towards human cervical cancer cells (HeLa).
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  17. DNA vaccine encoding avian influenza virus H5 and Esat-6 of Mycobacterium tuberculosis improved antibody responses against AIV in chickens
    Oveissi S, Omar AR, Yusoff K, Jahanshiri F, Hassan SS
    Comp Immunol Microbiol Infect Dis, 2010 Dec;33(6):491-503.
    PMID: 19781778 DOI: 10.1016/j.cimid.2009.08.004
    The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p<0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  18. Detection of pbp2b and ermB genes in clinical isolates of Streptococcus pneumoniae
    Kumari N, Navaratnam P, Sekaran SD
    J Infect Dev Ctries, 2008 Jun 01;2(3):193-9.
    PMID: 19738350
    BACKGROUND: Streptococcus pneumoniae is a major human pathogen. The emergence of penicillin resistant strains since the 1970s has been life threatening and the evolution of the bacteria have enabled itself to develop resistance to many other antibiotics such as the macrolides and the fluoroquinolones. This study aims to characterize S. pneumoniae isolates for the presence of penicillin and macrolide resistance genes.

    METHODOLOGY: One hundred and twenty clinical isolates of S. pneumoniae were obtained from patients of University Malaya Medical Centre (UMMC). The strains were screened using a multiplex real-time PCR method for the presence of alterations in the genes encoding the penicillin binding proteins: pbp2b, macrolide resistance determinant ermB and the pneumolysin gene, ply. Dual-labelled Taqman probes were used in the real-time detection method comprising three different genes labeled with individual fluorophores at different wavelengths. One hundred and twenty isolates from bacterial cultures and isolates directly from blood cultures samples were analyzed using this assay.

    RESULTS: A multiplex PCR comprising the antibiotic resistance genes, ermB and and pneumolysin gene (ply), a S. pneumoniae species specific gene, was developed to characterize strains of S. pneumoniae. Out of the 120 pneumococcal isolates, 58 strains were categorized as Penicillin Sensitive Streptococcus pneumoniae (PSSP), 36 as Penicillin Intermediate Streptococcus pneumoniae (PISP) and 26 as Penicillin Resistant Streptococcus pneumoniae (PRSP). All the 58 PSSP strains harboured the pbp2b gene while the 36 PISP and 26 PRSP strains did not harbour this gene, thus suggesting reduced susceptibility to penicillin. Resistance to erythromycin was observed in 47 of the pneumococcal strains while 15 and 58 were intermediate and sensitive to this drug respectively. Susceptibility testing to other beta-lactams (CTX and CRO) also showed reduced susceptibility among the strains within the PISP and PRSP groups but most PSSP strains were sensitive to other antibiotics.

    CONCLUSION: The characterization of pneumococcal isolates for penicillin and erythromycin resistance genes could be useful to predict the susceptibility of these isolates to other antibiotics, especially beta-lactams drugs. We have developed an assay with a shorter turnaround time to determine the species and resistance profile of Streptococcus pneumoniae with respect to penicillin and macrolides using the Real Time PCR format with fluorescent labeled Taqman probes, hence facilitating earlier and more definitive antimicrobial therapy which may lead to better patient management.

    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  19. Human nasal turbinates as a viable source of respiratory epithelial cells using co-culture system versus dispase-dissociation technique
    Noruddin NA, Saim AB, Chua KH, Idrus R
    Laryngoscope, 2007 Dec;117(12):2139-45.
    PMID: 17891046
    OBJECTIVE: To compare a co-culture system with a conventional dispase-dissociation method for obtaining functional human respiratory epithelial cells from the nasal turbinates for tissue engineering application.

    METHODS: Human respiratory epithelial cells were serially passaged using a co-culture system and a conventional dispase-dissociation technique. The growth kinetics and gene expression levels of the cultured respiratory epithelial cells were compared. Four genes were investigated, namely cytokeratin-18, a marker for ciliated and secretory epithelial cells; cytokeratin-14, a marker for basal epithelial cells; MKI67, a proliferation marker; and MUC5B, a marker for mucin secretion. Immunocytochemical analysis was performed using monoclonal antibodies against the high molecular-weight cytokeratin 34 beta E12, cytokeratin 18, and MUC5A to investigate the protein expression from cultured respiratory epithelial cells.

    RESULTS: Respiratory epithelial cells cultured using both methods maintained polygonal morphology throughout the passages. At passage 1, co-cultured respiratory epithelial showed a 2.6-times higher growth rate compared to conventional dispase dissociation technique, and 7.8 times higher at passage 2. Better basal gene expression was observed by co-cultured respiratory epithelial cells compared to dispase dissociated cells. Immunocytochemical analyses were positive for the respiratory epithelial cells cultured using both techniques.

    CONCLUSION: Co-culture system produced superior quality of cultured human respiratory epithelial cells from the nasal turbinates as compared to dispase dissociation technique.

    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
  20. Increased expression and hotspot mutations of the multidrug efflux transporter, CDR1 in azole-resistant Candida albicans isolates from vaginitis patients
    Looi CY, D' Silva EC, Seow HF, Rosli R, Ng KP, Chong PP
    FEMS Microbiol Lett, 2005 Aug 15;249(2):283-9.
    PMID: 16006060
    The aims of our research were to investigate the gene expression of the multidrug efflux transporter, CDR1 and the major drug facilitator superfamily transporter, MDR1 gene in azole drug-resistant Candida albicans and Candida glabrata clinical isolates recovered from vaginitis patients; and to identify hotspot mutations that may be present in the C. albicans CaCDR1 gene that could be associated with drug-resistance. The relative expression of the CDR1 and MDR1 transcripts in ketoconazole and clotrimazole-resistant isolates and drug-susceptible ATCC strains were determined by semi-quantitative reverse transcription-polymerase chain reaction. Expression of CaCDR1 transcript was upregulated to varying extents in all three azole-resistant C. albicans isolates studied (1.6-, 3.7- and 3.9-fold) and all three C. glabrata isolates tested (at 1.9-, 2.3- and 2.7-fold). The overexpression level of CaCDR1 in the isolates correlated with the degree of resistance as reflected by the minimum inhibitory concentration (MIC) of the drugs. The messenger RNA for another efflux pump, MDR1, was also overexpressed in one of the azole-resistant C. albicans isolates that overexpressed CDR1. This finding suggests that drug-resistance may involve synergy between energy-dependent drug efflux pumps CDR1p and MDR1p in some but not all isolates. Interestingly, DNA sequence analysis of the promoter region of the CaCDR1 gene revealed several point mutations in the resistant clinical isolates compared to the susceptible isolates at 39, 49 and 151 nucleotides upstream from the ATG start codon. This finding provides new information on point mutations in the promoter region which may be responsible for the overexpression of CDR1 in drug-resistant isolates.
    Matched MeSH terms: Reverse Transcriptase Polymerase Chain Reaction
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