Molecular bacteriology: a diagnostic tool for the millennium.
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Bacteria↗
Biomedical subjects
N A Saunders
Publications and source records attributed to N A Saunders.
Ventilatory decline after hypoxia and hypercapnia is not different between healthy young men and women.
The gradual decay in ventilation after removal of a respiratory stimulus has been proposed to protect against cyclic breathing disorders such as obstructive sleep apnea (OSA). The male predominance of OSA, and the increased incidence of OSA in women after menopause, indicates that the respiratory-stimulating effect of progesterone may provide protection against OSA by altering the rate of poststimulus ventilatory decline (PSVD). It was therefore hypothesized that PSVD is longer in premenopausal women than in men and is longer in the luteal menstrual phase compared with the follicular phase. PSVD was measured in 12 men and in 11 women at both their luteal and follicular phases, after cessation of isocapnic hypoxia and normoxic hypercapnia. PSVD was compared between genders and between women in the luteal and follicular phases by repeated-measures ANOVA. There were no significant differences in PSVD between any of the groups after either respiratory stimulus. This suggests that the higher occurrence of OSA in men does not reflect an underlying gender difference in PSVD and implies the increased prevalence of OSA in women after menopause is not representative of an effect of progesterone on PSVD.
Adult↗
Sustained isocapnic hypoxia suppresses the perception of the magnitude of inspiratory resistive loads.
The sensation of increased respiratory resistance or effort is likely to be important for the initiation of alerting or arousal responses, particularly in sleep. Hypoxia, through its central nervous system-depressant effects, may decrease the perceived magnitude of respiratory loads. To examine this, we measured the effect of isocapnic hypoxia on the ability of 10 normal, awake males (mean age = 24.0 +/- 1.8 yr) to magnitude-scale five externally applied inspiratory resistive loads (mean values from 7.5 to 54.4 cmH(2)O. l(-1). s). Each subject scaled the loads during 37 min of isocapnic hypoxia (inspired O(2) fraction = 0.09, arterial O(2) saturation of approximately 80%) and during 37 min of normoxia, using the method of open magnitude numerical scaling. Results were normalized by modulus equalization to allow between-subject comparisons. With the use of peak inspiratory pressure (PIP) as the measure of load stimulus magnitude, the perception of load magnitude (Psi) increased linearly with load and, averaged for all loaded breaths, was significantly lower during hypoxia than during normoxia (20.1 +/- 0.9 and 23.9 +/- 1.3 arbitrary units, respectively; P = 0. 048). Psi declined with time during hypoxia (P = 0.007) but not during normoxia (P = 0.361). Our result is remarkable because PIP was higher at all times during hypoxia than during normoxia, and previous studies have shown that an elevation in PIP results in increased Psi. We conclude that sustained isocapnic hypoxia causes a progressive suppression of the perception of the magnitude of inspiratory resistive loads in normal subjects and could, therefore, impair alerting or arousal responses to respiratory loading.
Adult↗
Regulation of the transglutaminase I gene. Identification of DNA elements involved in its transcriptional control in tracheobronchial epithelial cells.
The transglutaminase I (TGase I) gene encodes an enzyme that catalyzes the cross-linking of structural proteins involved in the formation of the cornified envelope during squamous cell differentiation. To identify DNA elements important for the transcriptional control of the TGase I gene, we analyzed the ability of a 2.9-kilobase pair (kb) upstream regulatory region to control the expression of a reporter gene in vivo and in vitro. Transgenic mice bearing the pTG(-2.9kb)CAT construct exhibited the same pattern of tissue-specific expression of CAT as reported for TGase I. Deletion analysis in transiently transfected rabbit tracheal epithelial cells indicated that two sequences from bp -490 to -470 and from -54 to -37 are involved in the activation of TGase I transcription. Point mutation analysis and mobility shift assays showed that the sequence located between -54 and -37 is a functional Sp1-like transcription element. Sp1 and Sp3, but not Sp2, are part of nuclear protein complexes from differentiated RbTE cells binding to this site. The element TGATGTCA between bp -490 and -470 is contained in a larger 22-bp palindrome and resembles the consensus cAMP response element-binding protein (CREB)/AP-1 element recognized by dimeric complexes of members of the CREB, ATF, Fos, and Jun families. Mutations in this sequence greatly reduced promoter activity. Supershift analysis identified CREB1, JunB, c-Fos, Fra-1, and c-Jun in protein complexes isolated from differentiated rabbit tracheal epithelial cells binding to this site. Our study shows that the Sp1- and CREB/AP-1-like sites act in concert to stimulate transcription of the TGase I gene. The 2.9-kb promoter region could guide expression of specific genes in the granular layer of the epidermis and could be useful in gene therapy.
Animals↗
Strain typing of Mycobacterium tuberculosis.
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Blotting, Southern↗
Cytochrome P450, CYP26AI, is expressed at low levels in human epidermal keratinocytes and is not retinoic acid-inducible.
Retinoids, and their synthetic analogues, are well-established regulators of the squamous differentiation programme both in vivo and in vitro. Despite this, very few studies have focused on the mechanism by which retinoid action is terminated, e.g. metabolism. Recently, a new cytochrome P450 family member (CYP26AI) was cloned. CYP26AI was reported to have substrate specificity for retinoids and to be retinoid-inducible. In this study, we have examined the expression and retinoic acid (RA) inducibility of CYP26AI in human epidermis and cultured keratinocytes. We found very low levels of CYP26AI mRNA expression in both epidermis and keratinocytes. Furthermore, we found no evidence for RA inducibility of CYP26 mRNA expression. This lack of RA inducibility was not due to inactivity of the retinoids, as we show that transglutaminase was still repressed by RA in the same cultures. Despite the low levels of CYP26AI expression in the keratinocytes, the keratinocytes were still capable of significant RA metabolism. In conclusion, our study reports, for the first time, that CYP26AI is unlikely to contribute to RA metabolism in keratinocytes. These studies also indicate that as yet unknown isoforms of cytochrome P450 may be involved in RA metabolism in keratinocytes.
Cell Line↗
Regulation of guanylate-binding protein expression in interferon-gamma-treated human epidermal keratinocytes and squamous cell carcinoma cells.
Interferon-gamma is a potent inducer of growth arrest and squamous differentiation of human epidermal keratinocytes in vitro. In order to understand the proximate events regulating interferon-gamma action we studied the relationship between interferon-gamma-mediated induction of a cytoplasmic guanylate-binding protein and the expression of growth and differentiation marker genes in normal and transformed keratinocytes. Induction of guanylate-binding protein mRNA by interferon-gamma was detectable at 4 h, was transcription dependent, and preceded changes in the expression of markers of growth arrest (E2F-1 mRNA downregulation) and differentiation (SQ37 mRNA induction). The Ec50 value for guanylate-binding protein induction (4 units interferon-gamma per ml) was lower than previously reported for SQ37 (40 units interferon-gamma per ml). Guanylate-binding protein mRNA appeared to be only moderately downregulated by modulators of the squamous phenotype such as retinoic acid and transforming growth factor-beta1. In addition, mRNA levels of E2F-1 or SQ37 were not altered in several squamous carcinoma cell lines treated with interferon-gamma. In contrast, guanylate-binding protein mRNA was highly induced in all these cell lines following interferon-gamma treatment. Further analysis of the signal transduction pathway mediating interferon-gamma responses using protein kinase inhibitors indicated that guanylate-binding protein induction in normal human epidermal keratinocyte cells was most likely protein kinase C independent. Our data suggest that more than one postreceptor interferon-gamma signaling pathway exists in keratinocytes and that at least one of these pathways is defective in squamous carcinoma cells. Furthermore, our data demonstrated that the failure of the squamous carcinoma cells to undergo interferon-gamma-induced growth arrest and differentiation is not due to an inherent defect in interferon-gamma receptor activation, but most likely is due to a defect in a non-guanylate-binding protein-dependent signaling pathway.
Carcinoma, Squamous Cell↗
Novel method for rapid determination of clarithromycin sensitivity in Helicobacter pylori.
A novel PCR-hybridization assay, performed in single closed capillaries, was developed to detect clarithromycin resistance-associated gene mutations in Helicobacter pylori. Mutations were detected by thermal analysis in 33 of 34 (97%) resistant isolates but not in 66 isolates determined to be sensitive by conventional antibiotic assays. The method was rapid and reproducible, and it reduced PCR product contamination risk.
Anti-Bacterial Agents↗
Daytime pulmonary hemodynamics in patients with obstructive sleep apnea without lung disease.
It is controversial whether obstructive sleep apnea (OSA) causes pulmonary hypertension (PH) in the absence of hypoxemic lung disease. To investigate this further we measured awake pulmonary hemodynamics, pulmonary gas exchange, and small airways function in 32 patients with OSA (apnea- hypopnea index, mean +/- SE, 46.2 +/- 3. 9/h) who had normal screening lung function. Pulmonary artery pressure (Ppa) and cardiac output were measured by Doppler echocardiography at three levels of inspired oxygen (FIO2 0.50, 0.21, and 0.11) and during incremental increases in pulmonary blood flow (10, 20, and 30 microgram/kg/min dobutamine infusions) while breathing 50% oxygen. Eleven patients had PH (mean Ppa >/= 20 mm Hg, Group I). They did not differ from patients without PH (Group II) in lung function, severity of sleep-disordered breathing, age, or body mass. Compared with Group II, Group I patients had increased small airways closure during tidal breathing (FRC-closing capacity: Group I, -0.16 +/- 0.11; Group II, 0.27 +/- 0.09 L; p < 0.05), more ventilation-perfusion inequality (AaPO2: 23.8 +/- 2.8; 19.8 +/- 1.4 mm Hg; p = 0.08), a greater pulmonary artery pressor response to hypoxia (DeltaPpa FIO2, 0.50 to 0.11: 16.4 +/- 1.93; 6.4 +/- 0.77 mm Hg; p < 0.05) and a marked rise in Ppa during increased pulmonary blood flow. We conclude that PH may develop in some patients with OSA without lung disease and that it is associated with small airways closure during tidal breathing and heightened pulmonary pressor responses to hypoxia and during increased pulmonary blood flow. Such changes are consistent with remodeling of the pulmonary vascular bed in affected patients with OSA, seemingly unrelated to severity of sleep-disordered breathing.
Blood Pressure↗
E2F1 messenger RNA is destabilized in response to a growth inhibitor in normal human keratinocytes but not in a squamous carcinoma cell line.
Keratinocyte growth arrest is characterized by a reduction in the activity and expression of E2F1. Here, we examine the role posttranscriptional processing plays in the down-regulation of E2F1 during keratinocyte growth arrest. E2F1 mRNA levels were undetectable within 8 h of exposure to the protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Assays of transcript stability indicated that, in untreated keratinocytes, the t 1/2 of E2F1 mRNA was 6.1 h and, in TPA-treated cells, it was 1.7 h. This destabilization was protein synthesis-dependent. In contrast, a growth inhibitor-resistant carcinoma cell line, SCC25, had a very stable E2F1 half-life that was only moderately reduced following TPA treatment. These data demonstrate that the initiation of keratinocyte growth arrest is associated with a rapid destabilization of E2F1 mRNA. These data are consistent with the proposition that inactivation of the posttranscriptional processing of important growth regulatory genes (e.g., E2F1) may contribute to neoplasia.
Blotting, Northern↗
Keratinocyte growth arrest is associated with activation of a transcriptional repressor element in the human cdk1 promoter.
In this study we examined the regulation of cdk1 expression in normal human epidermal keratinocytes (HEKs) and neoplastic keratinocytes. Keratinocytes were growth-arrested by allowing the cells to grow to confluence or by treating them with interferon-gamma (IFNgamma) or 12-O-tetradecanoyl phorbol-13-acetate (TPA). RT-PCR and Western blot analysis demonstrated that cdk1 was profoundly reduced in growth-arrested HEKs when compared with dividing HEKs. In contrast, a squamous carcinoma cell line, SCC25, did not growth-arrest in response to growth inhibitors and did not downregulate cdk1 expression. Transfection of HEKs with a reporter gene driven off a 2.5-kb fragment of the human cdk1 promoter indicated that the downregulation of cdkl upon growth arrest was transcriptional. Deletion mapping of the cdk1 promoter indicated that a repressor region was located between -949 - -722 bp. This repressor region was not operative in the SCC25 cells. Examination of DNA:protein binding complexes by gel-shift analysis indicated that nuclear factors from both proliferative and growth-arrested cells bound to the DNA fragment spanning -949- -722 bp. Further analysis revealed that this binding could be resolved into a constitutive and growth arrest-specific complex that bound in a similar fashion to regions spanning -892 - -831 bp and -831 - -774 bp, respectively. The putative growth arrest-specific complex was not found in contact-inhibited fibroblasts and was found at very low levels in SCC25 cells, indicating that the putative repressor binding was growth arrest-specific and possibly keratinocyte-specific. The binding complexes bound to these two fragments were localized, by competition analysis, to regions -874 - -853 bp and -830 - -800 bp. This is the first report of a transcriptional repressor being operative during keratinocyte growth arrest.
Base Sequence↗
Simplifying the molecular mechanisms of human papillomavirus.
Human papillomaviruses are a common human pathogen responsible for diseases varying in severity from warts to cervical cancer. This article examines the functions of the viral gene products and how they interact with cellular factors to replicate themselves and cause disease.
Antigens, Viral↗
Investigation of an outbreak of multidrug resistant tuberculosis among renal patients using rpo B gene sequencing and IS6110 inverse PCR.
A cluster of cases of tuberculosis among five patients receiving treatment for renal failure was investigated. Insertion sequence (IS6110) fingerprinting and antibiotic resistance profiling of the Mycobacterium tuberculosis isolates from four of the patients (A-D), who had been on the same ward, showed that three of these cases (A-C) were related, but that the fourth (D) was distinct. An isolate from the fifth patient (E), who had been on a separate ward, was indistinguishable from the outbreak strain by IS6110 profile. However, the isolate from patient E and a second isolate from patient A differed from the previous strains in being rifampicin resistant. Sequence analysis of the rpo B genes of the two rifampicin-resistant strains demonstrated the presence of different mutations, showing that they had evolved independently from the same source strain. IS6110 and rpo B gene analyses are invaluable for the accurate investigation of outbreaks of multidrug resistant tuberculosis.
Cross Infection↗
Ventilatory responses to sustained eucapnic hypoxia in healthy males during wakefulness and NREM sleep.
The effects of sustained eucapnic hypoxia (SEH, 20 minutes SaO2, approximately 80%) on ventilation and supraglottic airflow resistance (Rua) plus genioglossal (gg) and diaphragmatic (di) electromyograms (EMGs) were compared during wakefulness and nonrapid eye movement (NREM) sleep in six healthy normal male subjects. Early augmentation of ventilation was followed by decline or roll-off in both states. The augmentation of ventilation was less in sleep than wakefulness (e.g., after 5 minutes hypoxia, 140% and 167% of baseline, respectively, p < 0.05). This appeared to be due to three factors: 1) sleep-related increases in Rua [the ventilatory responses to SEH (sleep vs. awake) were inversely related to changes in Rua (sleep vs. awake) (p < 0.05)], 2. reduced central neural drive (inspiratory phasic EMG di after 5 minutes SEH, 111% and 121% of baseline, p < 0.05), and 3) failure to increase respiratory frequency during SEH sleep. There was also a nonsignificant trend to a biphasic response in EMG gg and a small increase in Rua during SEH.
Adult↗
E2F as a regulator of keratinocyte proliferation: implications for skin tumor development.
E2F and DP family members are established regulators of the cell cycle. In this study, we examined their activity/expression during keratinocyte growth arrest. Treating human epidermal keratinocytes with the growth inhibitors TPA or IFN-gamma or allowing the cells to reach confluence resulted in 90% inhibition of DNA synthesis, whereas a keratinocyte-derived squamous carcinoma cell line (SCC25) was resistant to growth inhibitors. Gel shift analysis of keratinocytes using an E2F response element indicated that growth arrest was associated with a decrease in all E2F binding complexes. This indicates that growth inhibition is not due to negative regulation by pocket proteins. Conversely, gel shift analysis of growth inhibitor-resistant SCC25 cells showed no decrease in E2F binding. If deregulated E2F expression/activity is involved in tumor development, then the deliberate deregulation of E2F activity may make keratinocytes resistant to growth inhibitors in much the same way as the SCC cells. The HPV16 E7 protein is known to activate E2F. Retroviral infection of keratinocytes with E7-expressing constructs resulted in growth inhibitor resistance, whereas infection with E6 constructs did not. E2F is a heterodimeric complex consisting of E2F family members (1-5) and DP proteins (1-3). Examination of the expression levels for E2F genes and other genes associated with the cell cycle indicated that E2F1 was profoundly decreased in growth-arrested keratinocytes (90%), whereas E2F3, E2F5, and DP1 were not. E2F2 and E2F4 were increased in IFN-gamma-treated keratinocytes but not in TPA-treated or confluent keratinocytes. In contrast, SCC25 cells did not undergo growth arrest and did not downregulate E2F1 mRNA expression in response to growth inhibitors. Our results indicate that E2F DNA binding and in particular E2F1 mRNA expression are associated with keratinocyte proliferation. Our results with the SCC25 cells and the E7-infected cells are consistent with the proposition that deregulated E2F expression/activity (in particular E2F1) may be involved in the unregulated proliferation of skin tumor cells.
Apoptosis↗
PCR-enzyme-linked immunosorbent assay and sequencing as an alternative to serology for M-antigen typing of Streptococcus pyogenes.
A rapid PCR-enzyme-linked immunosorbent assay for identification of 10 important emm gene types of Streptococcus pyogenes was developed. The emm genotypes of a coded panel of strains of known M serotype were determined, and in 144 of 149 cases (97%) the results were congruous. Strains of types that were not included in the panel of capture probes were emm genotyped by sequencing.
Antigens, Bacterial↗
Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism.
Mycobacterium tuberculosis isolates from 167 patients attending three London hospitals were analyzed by two techniques for strain differentiation. A significant number of isolates that appeared identical with the recently developed spoligotyping system could be distinguished from each other by IS6110 restriction fragment length polymorphism analysis, with the latter technique demonstrating a generally higher level of discrimination. Spoligotyping, on the other hand, was particularly useful for analysis of isolates with low IS6110 copy numbers, and use of the two techniques in tandem provided an optimal approach to the identification of clusters with epidemiological evidence consistent with recent transmission. Spoligotyping can be applied directly to clinical samples by PCR and provides an important tool for the rapid detection of nosocomial transmission of individual strains.
Adolescent↗
PCR-enzyme-linked immunosorbent assay and partial rRNA gene sequencing: a rational approach to identifying mycobacteria.
A PCR-enzyme-linked immunosorbent assay (ELISA) for amplification and rapid identification of mycobacterial DNA coding for 16S rRNA was developed. The PCR selectively targeted and amplified part of the 16S rRNA gene from all mycobacteria while simultaneously labelling one strand of the amplified product with a 5' fluorescein-labelled primer. The identity of the labelled strand was subsequently determined by hybridization to a panel of mycobacterial species-specific capture probes, which were immobilized via their 5' biotin ends to a streptavidin-coated microtiter plate. Specific hybridization of a 5' fluorescein-labelled strand to a species probe was detected colorimetrically with an anti-fluorescein enzyme conjugate. The assay was able to identify 10 Mycobacterium spp. A probe able to hybridize to all Mycobacterium species (All1) was also included. By a heminested PCR, the assay was sensitive enough to detect as little as 10 fg of DNA, which is equivalent to approximately three bacilli. The assay was able to detect and identify mycobacteria directly from sputa. The specificities of the capture probes were assessed by analysis of 60 mycobacterial strains corresponding to 18 species. Probes Avi1, Int1, Kan1, Xen1, Che1, For1, Mal1, Ter1, and Gor1 were specific. The probe Tbc1 cross-hybridized with the Mycobacterium terrae amplicon. Analysis of 35 strains tested blind resulted in 34 strains being correctly identified. This method could be used for rapid identification of early cultures and may be suitable for the detection and concurrent identification of mycobacteria within clinical specimens.
DNA Probes↗