ACTA PAEDIATRICA, Issue 2 2001
S Shang
Aim: The clinical diagnosis of sepsis is difficult, particularly in neonates. It is necessary to develop a rapid and reliable method for detecting bacteria in blood and cerebrospinal fluid (CSF) Polymerase chain reaction (PCR) and reverse hybridization of the 16S rRNA gene would permit fast and sensitive determination of the presence of bacteria and differentiate gram-positive bacteria from gram-negative ones in clinical specimens. Methods: We developed a pair of primers according to the gene encoding 16SrRNA found in all bacteria. DNA fragments from different bacterial species and from clinical samples were detected with PCR, and with reverse hybridization using a universal bacterial probe, a gram-positive probe and a gram-negative probe. Results: A 371 bp DNA fragment was amplified from 20 different bacterial species. No signal was observed when human DNA and viruses were used as templates. The sensitivity could be improved to 10T -12 g. All 26 culture-positive clinical samples (22 blood samples and 4 CSF samples) were positive with PCR. The gram-negative and gram-positive probes hybridized to clinical samples and to known bacterial controls, as predicted by Gram's stain characteristics. Conclusions: Our results suggest that the method of PCR and reverse hybridization is rapid, sensitive and specific in detecting bacterial infections. This finding may be significant in the clinical diagnosis of sepsis in neonates. [source]

Real-time primer design for DNA chips

CONCURRENCY AND COMPUTATION: PRACTICE & EXPERIENCE, Issue 9 2004
H. Simmler
Abstract The design of PCR or DNA chip experiments is a time-consuming process where bioinformatics is extensively used. The selection of the primers, which are immobilized on the DNA chip, requires a complex algorithm. Based on several parameters an optimized set of primers is automatically determined for a given gene sequence. This paper describes a parallel architecture which performs the optimization of the primer selection on a hardware accelerator. In contrast to the pure software approach, the parallel architecture gains a speedup of factor 500 using a PCI-based hardware accelerator. This approach allows an optimization of a specified primer set in real time. Copyright © 2004 John Wiley & Sons, Ltd. [source]

TRAF6 knockdown promotes survival and inhibits inflammatory response to lipopolysaccharides in rat primary renal proximal tubule cells

ACTA PHYSIOLOGICA, Issue 3 2010
S. Liu
Abstract Aim:, TRAF6 is a unique adaptor protein of the tumour necrosis factor receptor-associated factor family that mediates both tumour necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signalling. Activation of IL-1R/TLR and TNFR pathways in renal tubular cells contributes to renal injury. This study aimed to investigate if blockade of lipopolysaccharide (LPS)-triggered TLR4 signalling by small interfering RNA (siRNA) targeting TRAF6 protects survival and inhibits inflammatory response in isolated rat renal proximal tubular cells (PTCs). Methods:, PTCs isolated from F344 rat kidneys were transfected with chemically synthesized siRNA targeting TRAF6 mRNA. Real-time quantitative PCR was applied to measure mRNA level of TRAF6, TNF-,, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Protein levels of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, caspase 3 and cleaved caspase 3 were evaluated by Western blotting. Cell viability was analysed with XTT reagents. Results:, We found that the TRAF6 gene was effectively silenced in PTCs using siRNA. TRAF6 knockdown resulted in reduced TNF-, and IL-6 mRNA expression upon LPS challenge. LPS-induced phosphorylation of JNK and p38 was attenuated in TRAF6 siRNA-transfected cells while the change in the phosphorylation of ERK was not remarkable. TRAF6 knockdown was associated with increased cell viability and reduced protein level of cleaved caspase-3, both, in the absence and presence of LPS. Conclusion:, Our studies suggest that TRAF6 knockdown may inhibit inflammatory response and promote cell survival upon LPS challenge in primary rat proximal renal tubular cells. [source]

Characterization of sleep,wake patterns in a novel transgenic mouse line overexpressing human prepro-orexin/hypocretin

ACTA PHYSIOLOGICA, Issue 3 2010
K. A. Mäkelä
Abstract Aim:, Orexin/hypocretin peptides are expressed in the lateral hypothalamus and involved in the regulation of autonomic functions, energy homeostasis and arousal states. The sleep disorder narcolepsy, which is characterized by excessive daytime sleepiness and occurrence of sudden rapid eye movement (REM) sleep, is associated with a loss of orexin neurones. Our study investigated the effects of orexins on sleep,wake patterns in a novel transgenic mouse line overexpressing the human prepro-orexin (hPPO) gene under the control of its endogenous promoter. Methods:, Orexin overexpression was investigated by PCR, Southern and Western blotting as well as immunohistochemistry. Polysomnographic recordings were performed for analyses of sleep,wake patterns and for electroencephalographic activity during 24 h baseline and during and after 6 h of sleep deprivation (SD). Results:, Transgenic hPPO mice had increased expression of human prepro-orexin (hPPO) and orexin-A in the hypothalamus. Transgene expression decreased endogenous orexin-2 receptors but not orexin-1 receptors in the hypothalamus without affecting orexin receptor levels in the basal forebrain, cortex or hippocampus. Transgenic mice compared with their wild type littermates showed small but significant differences in the amount of waking and slow wave sleep, particularly during the light,dark transition periods, in addition to a slight reduction in REM sleep during baseline and during recovery sleep after SD. Conclusion:, The hPPO-overexpressing mice show a small reduction in REM sleep, in addition to differences in vigilance state amounts in the light/dark transition periods, but overall the sleep,wake patterns of hPPO-overexpressing mice do not significantly differ from their wild type littermates. [source]

Effects of short-term food deprivation on orexin-A-induced intestinal bicarbonate secretion in comparison with related secretagogues

ACTA PHYSIOLOGICA, Issue 3 2010
G. Flemström
Abstract Studies of gastrointestinal physiology in humans and intact animals are usually conducted after overnight fast. We compared the effects of orexin-A, vasoactive intestinal polypeptide (VIP), melatonin, serotonin, uroguanylin, ghrelin and prostaglandin E2 (PGE2) on duodenal bicarbonate secretion in fed and overnight fasted animals. This review is a summary of our findings. Secretagogues were administered by intra-arterial infusion or luminally (PGE2). Enterocyte intracellular calcium ([Ca2+]i) signalling was studied by fluorescence imaging. Total RNA was extracted, reverse transcripted to cDNA and expression of orexin receptors measured by quantitative real-time PCR. Orexin-A stimulates the duodenal secretion in continuously fed animals but not in food-deprived animals. Similarly, short-term fasting causes a 100-fold decrease in the amount of the muscarinic agonist bethanechol required for stimulation of secretion. In contrast, fasting does not affect secretory responses to intra-arterial VIP, melatonin, serotonin, uroguanylin and ghrelin, or that to luminal PGE2. Orexin-A induces [Ca2+]i signalling in enterocytes from fed rats but no significant [Ca2+]i responses occur in enterocytes from fasted animals. In addition, overnight fasting decreases the expression of mucosal orexin receptors. Short-term food deprivation thus decreases duodenal expression of orexin receptors and abolishes the secretory response to orexin-A as well as orexin-A-induced [Ca2+]i signalling. Fasting, furthermore, decreases mucosal sensitivity to bethanechol. The absence of declines in secretory responses to other secretagogues tested strongly suggests that short-term fasting does not affect the secretory capacity of the duodenal mucosa in general. Studies of intestinal secretion require particular evaluation with respect to feeding status. [source]

P28 Interleukin-8 from keratinocytes can be used to test for contact allergy

CONTACT DERMATITIS, Issue 3 2004
Bolli Bjarnason
Objective:, To investigate whether secretion of interleukin-8 (IL-8) proteins by keratinocytes following in vitro exposure to a contact allergen can be used to detect contact allergy. Methods:, Suction blisters were made on skin of allergic and anergic subjects to urushiol, the contact allergen of poison ivy. Keratinocyte cultures were prepared and exposed to the allergen in vitro. Controls were the allergen solvent. Variable allergen concentrations, allergen exposure times and cell culture times were used. At the end of each culture time, IL-8 RNA and protein of the culture supernatants were analyzed by PCR and ELISA. Results:, The concentration of IL-8 in the supernatants proved to be a successful way to distinguish between subjects who patch tested positive with a non-toxic concentration of urushiol and subjects who tested negative. In the allergic subjects, a correlation was established between the dose of the allergen and the IL-8 protein concentration in the supernatants. Conclusions:, In vitro testing of contact allergies in patients makes possible an objective assessment of their allergic status without causing a booster effect or risking active sensitizations. The results indicate that the method may be used as an alternative method to animal models for testing consumer products before their marketing, thus avoiding ethical problems and problems related to interpretation of tests because of biological differences between animals and humans. [source]

Oestradiol and SERM treatments influence oestrogen receptor coregulator gene expression in human skeletal muscle cells

ACTA PHYSIOLOGICA, Issue 3 2009
C. M. Dieli-Conwright
Abstract Aim:, Oestrogen receptors (ER) are present in human skeletal muscle (hSkM) cells; however, the function of the receptor is currently unknown. We investigated the influence of oestradiol and selective ER modulators [tamoxifen (TAM), raloxifene (RAL)] on ER coregulator mRNA expression in hSkM. Methods:, Human skeletal muscle cells were treated with 10 nm oestradiol, 5 ,m TAM and 10 ,m RAL over a 24-h period. Following the treatment period, mRNA expression was quantified using real-time PCR to detect changes in ER-,, ER-,, steroid receptor coactivator (SRC), silencing mediator for retinoid and thyroid hormone receptors (SMRT), MyoD, GLUT4 and c-fos. Results:, ER-, mRNA expression increased with all three drug treatments (P < 0.05) while there was no change in mRNA expression of ER-, in hSkM cells. mRNA expression of SRC increased and SMRT decreased with oestradiol, TAM and RAL in hSkM cells (P < 0.05). Importantly, mRNA expression of MyoD increased with oestradiol and decreased with TAM and RAL in hSkM cells (P < 0.05). mRNA expression of GLUT4 increased with oestradiol and RAL and decreased with TAM in hSkM cells (P < 0.05). Conclusions:, These findings are novel in that they provide the first evidence that oestradiol and selective ER modulators influence ER-, function in hSkM cells. This demonstrates the importance of the ER and alterations in its coregulators, to potentially prevent sarcopenia and promote muscle growth in postmenopausal women using these forms of hormone replacement therapy. [source]

Enhanced pulmonary expression of the TrkB neurotrophin receptor in hypoxic rats is associated with increased acetylcholine-induced airway contractility

ACTA PHYSIOLOGICA, Issue 3 2009
L. K. Sciesielski
Abstract Aim:, We have recently reported that hypoxia stimulates transcription of the TrkB neurotrophin receptor in cultured cells via stabilization of hypoxia-inducible factor-1,. Here we investigated whether the expression of TrkB and other neurotrophin receptors is oxygen-sensitive also in vivo, and explored the functional consequences of an oxygen-regulated TrkB expression. Methods:, Rats were exposed either to 21% O2 or 8% O2 for 6 h and TrkB was analysed by reverse transcription real-time PCR, in situ mRNA hybridization, and immunological techniques. The importance of the brain-derived neurotrophic factor (BDNF)-TrkB pathway in the control of mechanical airway function was assessed on isolated tracheal segments from normoxic and hypoxic rats. Results:,TrkB transcripts were increased approx. 15-fold in the lungs of hypoxic rats, and the respiratory epithelium was identified as the site of enhanced TrkB expression in hypoxia. The TrkB ligand, BDNF, significantly increased the contractile response to acetylcholine (ACh) of isolated tracheal segments from hypoxic but not from normoxic rats. This effect of BDNF was prevented by pre-incubation of the tissue specimens with the tyrosine kinase inhibitor K252a and by mechanical removal of the TrkB containing airway epithelium. Likewise, the nitric oxide (NO) synthase inhibitor l -NAME abrogated the influence of BDNF on ACh-induced contractions of isolated tracheal segments from hypoxic rats. Conclusion:, These results demonstrate that systemic hypoxia stimulates expression of the TrkB neurotrophin receptor in the airway epithelium. Furthermore, activation of TrkB signalling by BDNF in hypoxia enhances mechanical airway contractility to ACh through a mechanism that requires NO. [source]

Purinergic activation of a leak potassium current in freshly dissociated myocytes from mouse thoracic aorta

ACTA PHYSIOLOGICA, Issue 2 2009
S. Hayoz
Abstract Aim:, Exogenous ATP elicits a delayed calcium-independent K+ current on freshly isolated mouse thoracic aorta myocytes. We investigated the receptor, the intracellular pathway and the nature of this current. Methods:, The patch-clamp technique was used to record ATP-elicited delayed K+ current in freshly dissociated myocytes. Results:, ATP-elicited delayed K+ current was not inhibited by a ,cocktail' of K+ channel blockers (4-AP, TEA, apamin, charybdotoxin, glibenclamide). The amplitude of the delayed K+ current decreased after the reduction of extracellular pH from 7.4 to 6.5. These two characteristics suggest that this current could be carried by the TASK subfamily of ,twin-pore potassium channels' (K2P). Purinergic agonists including dATP, but not ADP, activated the delayed K+ current, indicating that P2Y11 is the likely receptor involved in its activation. The PKC activator phorbol ester 12,13-didecanoate stimulated this current. In addition, the PKC inhibitor Gö 6850 partially inhibited it. Real-time quantitative PCR showed that the genes encoding TASK-1 and TASK-2 are expressed. Conclusion:, Our results indicate that blocker cocktail-insensitive delayed K+ current in freshly dissociated aortic myocytes is probably carried by the TASK subfamily of twin-pore channels. [source]

HLA-B27 typing: Evaluation of an allele-specific PCR melting assay and two flow cytometric antigen assays

CYTOMETRY, Issue 1 2005
Michael T. Seipp
Abstract Background Human leukocyte antigen B27 (HLA-B27) is a major histocompatibility complex class 1 molecule that is strongly associated with the disease ankylosing spondylitis. Testing for HLA-B27 is of diagnostic value because 90% of patients with ankylosing spondylitis have the B27 antigen. Two commonly used HLA-B27 flow cytometric assays are commercially available. Methods An allele-specific polymerase chain reaction (PCR) melting assay for HLA-B27 was compared with two available antigen assays on 371 clinical samples. The accuracy of the assays was measured by receiver operating characteristic analysis using the PCR method and sequencing as the reference standard. Results When PCR results were compared with those of the antigen assays, complete concordance was observed except for five discrepant results that were resolved by sequence analysis. Using DNA sequencing as the gold standard, the sensitivity and specificity of PCR were 99.6 and 100.0, those of the best single antigen assay were 98.2 and 97.6, and those of a reflex combination of both antigen assays were 98.8 and 97.6. Conclusions The allele-specific PCR melting assay for HLA-B27 genotyping is easy to perform and has better sensitivity and specificity than antigen assays. The performance of the two flow cytometric antigen assays depends on the antibody used and the positive cutoff values assigned. © 2004 Wiley-Liss, Inc. [source]

Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipients

CYTOPATHOLOGY, Issue 6 2008
M. Koukoulaki
Objective:, BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients. Patients and methods:, We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood. Results:, Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR. Conclusions:, Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation. [source]

Oestrogen receptor , is expressed in adult human skeletal muscle both at the mRNA and protein level

ACTA PHYSIOLOGICA, Issue 4 2003
A. Wiik
Abstract Aim:, There are two known oestrogen receptors (ER), oestrogen receptor , (ER,) and the recently cloned oestrogen receptor , (ER,). ER, mRNA has been detected in mouse, rat, bovine and human skeletal muscle. ER, mRNA has been detected in bovine skeletal muscle. To our knowledge, no study has investigated the expression of oestrogen receptor , in human skeletal muscle. Therefore, the primary aim of the present investigation was to study ER, mRNA and protein expression in human skeletal muscle. In addition the ER, expression was also studied. Methods:, Muscle biopsies were taken from vastus lateralis in six healthy adults (three women and three men). mRNA expression was detected with real-time PCR (TaqMan) and protein localization by immunohistochemistry. Results:, A clear expression of ER, and ER, mRNA was seen in skeletal muscle in all subjects. The ER, mRNA expression was 180 fold higher compared with that of ER, mRNA. Immunohistochemistry demonstrated positive staining for ER,, but not for ER,, with localization to the nuclei of skeletal muscle fibres. On average, 70% of all nuclei were ER, -positive. Conclusion:, The present study shows for the first time ER, mRNA and protein expression in human skeletal muscle tissue in both males and females. [source]

Trials update in wales

CYTOPATHOLOGY, Issue 2007
A. Fiander
Three ongoing studies will be presented and discussed. Prevalence of Human Papillomavirus Infection in a South Wales Screening population Methods: A total of 10 000 consecutive, anonymous liquid based cytology screening samples were collected over a five month period in 2004. Age, cytology result and social deprivation score was provided for each specimen. The methodology was chosen to ensure inclusion of all women attending routine cervical screening, avoiding potential constraints associated with obtaining individual informed consent. The liquid based cytology samples were processed and reported by the receiving cytology laboratory and the residual specimens sent to the HPV Research Laboratory, Wales College of Medicine, where they were processed and stored at -80°C until analysis. High risk and low risk HPV Typing was undertaken using PCR , EIA (Jacobs et al 1997). Full high risk typing was performed on HPV positive specimens. Results: The study population had a mean age of 38 years with 92% negative, 5% borderline and 3% dyskaryotic cytology. The average social deprivation score was 17.4 (based upon the Welsh Index of multiple deprivation). The following results will be presented: HPV prevalence by age. HPV prevalence by cytology result. Type specific HPV prevalence in single and multiple infection. Conclusion: This study represents the largest type specific HPV Prevalence Study in the UK to date. As such it will form a useful base line against which to access performance of marketed HPV tests and evaluating the impact following implementation of HPV vaccination. [Funded by Welsh Office for Research and Development] CRISP , 1 Study (Cervical Randomized Intervention Study Protocol -1) Background: Indole-3-carbinol (I3C) and Diindolylmethane (DIM) are found in cruciferous vegetables and have been identified as compounds that could potentially prevent or halt carcinogenesis. I3C spontaneously forms DIM in vivo during acid digestion. I3C has been shown to prevent the development of cervical cancer in HPV 16 transgenic mice and both I3C and DIM have been shown to promote cell death in cervical cancer cell models. DIM is the major active bi-product of I3C and preliminary data indicate that DIM is active in cervical dysplasia and may be better tolerated than I3C. Aim: To investigate chemoprevention of high grade cervical neoplasia using Diindolylmethane (DIM) supplementation in women with low grade cytological abnormalities on cervical cytology. Objectives: To observe any reduction in the prevalence of histological proven high-grade cervical intraepithelial neoplasia (CIN) after 6 months of supplementation. ,,To observe any reduction in the prevalence of cytological abnormalities. ,,To observe any changes in the clinical appearance of the cervix. To assess acceptability and monitor any side effects of DIM supplementation. ,,To assess whether any benefit is seen in relation to Human Papillomavirus (HPV) status including HPV Type, Viral load and integration. Methods: This is a double blind randomized placebo-controlled trial involving 600,700 women with low grade cytological abnormalities on a cervical smear. Randomization is in the ratio of 2 : 1 in favour of active medication. Women with first mildly dyskaryotic smear or second borderline smear are eligible. They are asked to take two capsules daily for 6 months. At the end of 6 months they undergo repeat cervical cytology, HPV testing and colposcopy. Results: A progress report will be given for this ongoing study. [Funded: - Cancer Research UK] Type Specific HPV Infection in Welsh Cervical Cancers Background: Whilst there have been numerous studies of HPV infection associated with cervical cancer and on prevalence of Human Papillomavirus in diverse populations there have been no studies of these variables in the same population. Against a background of prophylactic HPV vaccination it is important to assess potential protection against cervical cancer within a given population. The most comprehensive analysis of HPV type specific cervical cancer is a meta-analysis published by the IARC in 2003. This however included only three UK based studies, totalling 118 cases, 75 of which were only investigated by HPV type PCR for four high risk types. None of this data was presented with associated population based prevalence data. Therefore, the research objectives for this study in combination with the first study above, are as follows: To determine the frequency of specific HPV types in cervical cancers in Wales. To compare the distribution of specific HPV types amongst cervical cancers with their prevalence in the general population. This will allow accurate delineation of the relationship between prevalence of specific HPV types in the general population and their association with clinically relevant disease. This information is a pre-requisite to assess the potential impact of prophylactic vaccination against HPV infection in Wales. Methods: Welsh Cervical Cancer specimens from 2000,2005 will be identified from pathology departments within Wales. The pathology of each tumour will be reviewed by a single Gynaecological Pathologist. The age of the patient and pathological features of the tumour will be noted. DNA will be extracted from the paraffin sections and HPV typed by PCR-EIA. Results: A progress report will be given for this ongoing study. [Funded by Welsh Office for Research and Development] [source]

Role of ancillary techniques in diagnosing and subclassifying non-Hodgkin's lymphomas on fine needle aspiration cytology

CYTOPATHOLOGY, Issue 5 2006
P. DeyArticle first published online: 8 SEP 200
Non-Hodgkin's lymphomas (NHL) are tumours of the lymphoid cells. During the process of development of lymphoid cells, neoplasia may evolve at any point. Neoplastic cells usually carry the imprint of cell of origin at the stage of origin. Various types of NHL may have similar morphology with wide variation in origin, immunophenotype and other biological features. Different ancillary laboratory techniques may help to overcome the limitations of morphology in this aspect. The commonly used ancillary techniques in lymphomas are immunocytochemistry (IC), flow cytometry, Southern blot (SB) technique, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH). In addition, laser scanning cytometry (LSC) and DNA microarray technologies are in the research phase. Various laboratory techniques are used for immunophenotyping, demonstration of monoclonality, identification of chromosomal translocation, assessment of cell kinetics and expression of mRNA in the tumour cells. Flow cytometry helps in rapid immunophenotying of NHL and it has an added advantage over IC in recognizing the co-expression of CD markers. Fine needle aspiration cytology (FNAC) combined with flow immunophenotyping may help us to diagnose and subclassify certain NHLs, such as follicular lymphoma and mantle cell lymphoma, which were previously recognized as pure morphological entities. Loss of morphology is one of the important limitations of flow cytometry. LSC can overcome this limitation by studying morphology along with the immunophenotyping pattern of individual cells. Chromosomal changes in NHL can be identified by SB, PCR and FISH. Molecular diagnosis of NHL helps in diagnosis, subclassification, prognostic assessment and even in planning of therapy. DNA microarray is a relatively newer and promising technology. It gives information about the expression of several thousands of genes in a tumour in a single experiment. In the near future, FNAC combined with ancillary techniques may play a major role in diagnosis, subclassification and management of lymphomas. [source]

Significance of high-risk human papillomavirus detection by polymerase chain reaction in primary cervical cancer screening

CYTOPATHOLOGY, Issue 2 2001
Y. L. Oh
Significance of high-risk human papillomavirus detection by polymerase chain reaction in primary cervical cancer screening The purposes of this study were to evaluate the incidence of high-risk human papillomavirus (HPV) infection by polymerase chain reaction (PCR) and to assess its diagnostic usefulness in primary cervical screening. PCR testing for HPV type 16, 18, 31 and 33 was performed on 1305 specimens obtained during routine cervical cancer screening. We analysed the concurrent cervical smears and biopsy, and correlated them with the HPV infection status. We also evaluated histologically-proven cases with ASCUS smears according to HPV infection. HPV DNA was identified in eight (0.7%) of 1144 cytologically normal patients; nine (10.5%) of 86 ASCUS; seven (25.0%) of 28 LSIL; 26 (78.8%) of 33 HSIL; and in all of three squamous cell carcinomas (SCC). HPV positivity was significantly associated with cytohistological diagnosis for HSIL of more. In addition, HPV-positive ASCUS cases were found to be associated with histological abnormality rather than HPV-negative. The results indicate that high-risk HPV testing by PCR could be a useful adjunct tool for Pap smear in primary cervical screening. The combination of Pap smear and high-risk HPV testing by PCR might reduce unnecessary colposcopy-guided biopsy of women with cytological diagnosis of ASCUS. [source]

The effect of combined simulated microgravity and microgrooved surface topography on fibroblasts

CYTOSKELETON, Issue 3 2007
W. A. Loesberg
Abstract This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 ,m, width: 1, 2, 5, and 10 ,m), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment and area. Confocal laser scanning microscopy visualised distribution of actin filaments and focal adhesion points. Finally, expression of collagen type I, fibronectin, and ,1- and ,1-integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces decreased under simulated microgravity, especially after 24 h of culturing. Cell surface area on grooved substrata were significantly smaller than on smooth substrata, but simulated microgravity on the grooved groups resulted in an enlargement of cell area. ANOVA was performed on all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, gene levels were reduced by microgravity particularly those of ,1-integrin and fibronectin. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by microgravity conditions. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Dynein light chain family in Tetrahymena thermophila

CYTOSKELETON, Issue 2 2007
David E. Wilkes
Abstract Dyneins are large protein complexes that produce directed movement on microtubules. In situ, dyneins comprise combinations of heavy, intermediate, light-intermediate, and light chains. The light chains regulate the locations and activities of dyneins but their functions are not completely understood. We have searched the recently sequenced Tetrahymena thermophila macronuclear genome to describe the entire family of dynein light chains expressed in this organism. We identified fourteen genes encoding putative dynein light chains and seven genes encoding light chain-like proteins. RNA-directed PCR revealed that all 21 genes were expressed. Quantitative real time reverse transcription PCR showed that many of these genes were upregulated after deciliation, indicating that these proteins are present in cilia. Using the nomenclature developed in Chlamydomonas, Tetrahymena expresses two isoforms each of LC2, LC4, LC7, and Tctex1, three isoforms of p28, and six LC8/LC8-like isoforms. Tetrahymena also expresses two LC3-like genes. No Tetrahymena orthologue was found for Chlamydomonas LC5 or LC6. This study provides a complete description of the different genes and isoforms of the dynein light chains that are expressed in Tetrahymena, a model organism in which the targeted manipulation of genes is straightforward. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source]

The effect of combined hypergravity and microgrooved surface topography on the behaviour of fibroblasts

CYTOSKELETON, Issue 7 2006
W. A. Loesberg
Abstract This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 1 ,m, width: 1, 2, 5, 10 ,m), which undergo artificial hypergravity by centrifugation (10, 24 and 50 g; or 1 g control). The aim of the study was to clarify which of these parameters was more important to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell spreading and alignment. Confocal laser scanning microscopy visualised distribution of actin filaments and vinculin anchoring points through immunostaining. Finally, expression of collagen type I, fibronectin, and ,1 - and ,1 -integrin were investigated by PCR. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata (control), cells spread out in a random fashion. The alignment of cells cultured on grooved surfaces increased with higher g-forces until a peak value at 25 g. An ANOVA was performed on the data, for all main parameters: topography, gravity force, and time. In this analysis, all parameters proved significant. In addition, most gene levels were reduced by hypergravity. Still, collagen type 1 and fibronectin are seemingly unaffected by time or force. From our data it is concluded that the fibroblasts primarily adjust their shape according to morphological environmental cues like substratum surface whilst a secondary, but significant, role is played by hypergravity forces. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source]

Human Papillomavirus and Overexpression of P16INK4a in Nonmelanoma Skin Cancer

DERMATOLOGIC SURGERY, Issue 3 2004
Ingo Nindl PhD
Background. P16INK4a overexpression has been identified as a specific biomarker in high-risk human papillomavirus (HPV),infected cervical (pre)cancer lesions. Objective. To evaluate the overexpression of this cyclin-dependent kinase inhibitor in skin tumors depending on HPV infections, we analyzed normal skin, benign skin disease, and skin cancer specimens. Methods. Biopsies of 23 patients with normal histology (3), psoriasis (2), verrucae vulgaris (2), actinic keratoses (5), squamous cell carcinoma (SCC) in situ (3), Bowen's carcinoma (1), and SCC (7) were analyzed. Specimens of 23 patients were immunostained using the monoclonal antibody E6H4 specific for p16INK4a. HPV status was assessed by a polymerase chain reaction (PCR) system to detect all currently known HPV types. MY (MY09/MY11 and MYN9/MYN10)-, CP (CP65/CP70 and CP66/CP69)-nested PCR, and three single PCR methods CN1, CN3, and CN4 were used in a first step, and HPV typing was performed by restriction fragment length polymorphism analysis. Only ,-globin,positive patients were included in this study. Results. HPV DNA was detected in all actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC, in 50% (one of two) of verrucae vulgaris, in 66% (two of three) of normal skin, and in none of two psoriasis. P16INK4a expression was not detected in normal skin, psoriasis, and verrucae vulgares. Overexpression of p16INK4a was detected in a subset of dysplastic cells (10% to 80%) of all skin (pre)cancer lesions such as actinic keratoses, SCC in situ, Bowen's carcinoma, and SCC infected with HPV independent of sun exposure. Conclusion. P16INK4a appears to be overexpressed in a portion of dysplastic cells from actinic keratoses and SCC. Further studies to examine the association of HPV infection and the overexpression of p16INK4a are warranted. [source]

Single-cell gene profiling of planarian stem cells using fluorescent activated cell sorting and its "index sorting" function for stem cell research

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2010
Tetsutaro Hayashi
To achieve an integrated understanding of the stem cell system of planarians at both the cellular and molecular levels, we developed a new method by combining "fluorescent activated cell sorting (FACS) index sorting" analysis and single-cell reverse transcription,polymerase chain reaction (RT,PCR) to detect the gene expression and cell cycle state of stem cells simultaneously. Single cells were collected using FACS, and cDNAs of each cell were used for semi-quantitative RT,PCR. The results were plotted on the FACS sorting profile using the "index sorting" function, which enabled us to analyze the gene expression in combination with cell biological data (such as cell cycle phase) for each cell. Here we investigated the adult stem cells of planarians using this method and obtained findings suggesting that the stem cells might undergo commitment during S to G2/M phase. This method could be a powerful and straightforward tool for examining the stem cell biology of not only planarians but also other organisms, including vertebrates. [source]

Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner ear

DEVELOPMENTAL DYNAMICS, Issue 4 2004
John A. Germiller
Abstract Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors,and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation. Developmental Dynamics 231:815,827, 2004. © 2004 Wiley-Liss, Inc. [source]

Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival

DEVELOPMENTAL NEUROBIOLOGY, Issue 13 2009
Laura Lossi
Abstract Apoptosis can be modulated by K+ and Ca2+ inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca2+ signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K+/Ca2+ homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K+]e and [Ca2+]i to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K+]e under conditions of immaturity, is independent of extracellular Ca2+ and operates via IP3 channels. The second leads to influx of extracellular Ca2+ following activation of ryanodine channels in the presence of physiological [K+]e, when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source]

Time course analysis of gene expression during light-induced photoreceptor cell death and regeneration in albino zebrafish

DEVELOPMENTAL NEUROBIOLOGY, Issue 8 2007
Sean C. Kassen
Abstract Constant intense light causes apoptosis of rod and cone photoreceptors in adult albino zebrafish. The photoreceptors subsequently regenerate from proliferating inner nuclear layer (INL) progenitor cells that migrate to the outer nuclear layer (ONL) and differentiate into rods and cones. To identify gene expression changes during this photoreceptor regeneration response, a microarray analysis was performed at five time points during the light treatment. The time course included an early time point during photoreceptor death (16 h), later time points during progenitor cell proliferation and migration (31, 51, and 68 h) and a 96 h time point, which likely corresponds to the initial photoreceptor differentiation. Mean expression values for each gene were calculated at each time point relative to the control (0 h light exposure) and statistical analysis by one-way ANOVA identified 4567 genes exhibiting significant changes in gene expression along the time course. The genes within this data set were clustered based on their temporal expression patterns and proposed functions. Quantitative real-time PCR validated the microarray expression profiles for selected genes, including stat3 whose expression increased markedly during the light exposure. Based on immunoblots, both total and activated Stat3 protein expression also increased during the light treatment. Immunolocalization of Stat3 on retinal tissue sections demonstrated increased expression in photoreceptors and Müller glia by 16 h of light exposure. Some of the Stat3-positive Müller cells expressed PCNA at 31 h, suggesting that Stat3 may play a role in signaling a subset of Müller cells to proliferate during the regeneration response. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source]

N-cadherin is regulated by gonadal steroids in adult sexually dimorphic spinal motoneurons

DEVELOPMENTAL NEUROBIOLOGY, Issue 4 2001
Douglas A. Monks
Abstract Gonadal steroids influence the morphology and function of neurons in the adult spinal cord through cellular and molecular mechanisms that are largely unknown. The cadherins are cell adhesion molecules that participate in the formation and organization of the CNS during embryonic development, and recent evidence suggests that the cadherins continue to regulate neural structure and function in adulthood. Using degenerate oligonucleotides coding conserved regions of the catenin-binding domain of classical cadherins in a RT-PCR cloning strategy, we identified several cadherin subtypes, the most frequently cloned being N-, E-, and R-cadherin, suggesting that these are the major classical cadherin subtypes present in the adult male rat lumbosacral spinal cord. We then examined cadherin expression levels of these cadherin subtypes under steroid conditions known to induce plastic changes in spinal motoneurons. Semiquantitative PCR revealed that mRNA levels of N-cadherin, but not E-cadherin or R-cadherin, are elevated in castrated rats treated with testosterone, 17,-estradiol, or dihydrotestosterone relative to castrate rats not treated with steroids. Immunolocalization of N-cadherin revealed that steroid treatment increased N-cadherin expression levels in functionally related neural populations whose morphology and function are regulated by steroids. These results suggest a role for N-cadherin in steroid-induced neuroplastic change in the adult lumbar spinal cord. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 255,264, 2001 [source]

Effect of chronic denervation and denervation-reinnervation on cytoplasmic creatine kinase transcript accumulation

DEVELOPMENTAL NEUROBIOLOGY, Issue 3 2001
Charles H. Washabaugh
Abstract The extensor digitorum longus (EDL) and soleus muscles of adult mice were chronically denervated or denervated and allowed to reinnervate. Muscles were evaluated 1, 5, 14, 21, and 52 days after sciaticectomy. In terms of weight loss, myofiber atrophy, degeneration, and fibrosis, the soleus muscle was more affected than the EDL by chronic denervation. Fifty-two days after chronic denervation, the number of molecules of MCK/ng total RNA in both muscles (determined with competitive PCR) decreased, with the soleus muscle being more affected. At that stage, BCK mRNA levels in the denervated soleus were unchanged, but they were increased (>50%) in the EDL. Reinnervation restored MCK transcript accumulation in the EDL, whereas, in the soleus MCK, transcripts exceeded control values by 57%, approaching levels in the reinnervated EDL. Despite restoration of MCK mRNA levels, the number of molecules of BCK mRNA/ng total RNA was four- to fivefold higher in reinnervated versus control muscles, suggesting that the genes encoding the CK mRNAs are not coordinately regulated in adult muscle. The role of denervation induced, fiber type changes in regulating CK mRNA accumulation has been evaluated. Electron microscopic analyses have established that fibrosis is not a factor that determines BCK mRNA levels in the chronically denervated or denervated-reinnervated muscles. CK isozyme analyses support the hypothesis that a greater proportion of BCK mRNA found in 52 day chronically denervated and denervated-reinnervated muscles is produced in myofibers vs. nonmuscle cells than in control muscles. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 194,206, 2001 [source]

Lack of association between serum adiponectin levels and the Pro12Ala polymorphism in Asian Indians

DIABETIC MEDICINE, Issue 4 2007
V. Radha
Abstract Aims, The aim of the study was to investigate the association of serum adiponectin levels with the Pro12Ala polymorphism of the peroxisome proliferator activated receptor-, (PPARG) gene in Asian Indians. Methods, We selected 400 diabetic subjects, 200 with the Pro12Pro genotype (100 male and 100 female) and 200 with the Pro12Ala genotype (100 male and 100 female) and 400 age- and sex-matched normal glucose tolerance subjects with similar genotype profiles from the Chennai Urban Rural Epidemiology Study. Fasting serum adiponection levels were measured using radioimmunoassay. The Pro12Ala polymorphism was genotyped by PCR,restriction fragment length polymorphism using BstUI. Results, All clinical and biochemical parameters were similar in the subjects with the Pro12Pro and Pro12Ala genotypes. There was no significant difference in serum adiponectin values between subjects with the Pro12Pro and Pro12Ala genotypes (males 5.4 vs. 5.8 µg/ml, P = 0.546; females 6.9 vs. 7.2 µg/ml, P = 0.748). Adiponectin values did not differ among these two genotypes even when categorized based on their diabetes status (normal glucose tolerance Pro12Pro 7.9 vs. Pro12Ala 7.7 µg/ml, P = 0.994; diabetes Pro12Pro 4.7 vs. Pro12Ala 5.4 µg/ml, P = 0.622). Conclusion, The Pro12Ala polymorphism of the PPARG gene is not associated with serum adiponectin levels in Asian Indians. [source]

Peroxisome proliferator-activated receptor-, co-activator-1, (PGC-1,) gene polymorphisms and their relationship to Type 2 diabetes in Asian Indians

DIABETIC MEDICINE, Issue 11 2005
K. S. Vimaleswaran
Abstract Aims The objective of the present investigation was to examine the relationship of three polymorphisms, Thr394Thr, Gly482Ser and +A2962G, of the peroxisome proliferator activated receptor-, co-activator-1 alpha (PGC-1,) gene with Type 2 diabetes in Asian Indians. Methods The study group comprised 515 Type 2 diabetic and 882 normal glucose tolerant subjects chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The three polymorphisms were genotyped using polymerase chain reaction,restriction fragment length polymorphism (PCR,RFLP). Haplotype frequencies were estimated using an expectation,maximization (EM) algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. Results The three polymorphisms studied were not in linkage disequilibrium. With respect to the Thr394Thr polymorphism, 20% of the Type 2 diabetic patients (103/515) had the GA genotype compared with 12% of the normal glucose tolerance (NGT) subjects (108/882) (P = 0.0004). The frequency of the A allele was also higher in Type 2 diabetic subjects (0.11) compared with NGT subjects (0.07) (P = 0.002). Regression analysis revealed the odds ratio for Type 2 diabetes for the susceptible genotype (XA) to be 1.683 (95% confidence intervals: 1.264,2.241, P = 0.0004). Age adjusted glycated haemoglobin (P = 0.003), serum cholesterol (P = 0.001) and low-density lipoprotein (LDL) cholesterol (P = 0.001) levels and systolic blood pressure (P = 0.001) were higher in the NGT subjects with the XA genotype compared with GG genotype. There were no differences in genotype or allelic distribution between the Type 2 diabetic and NGT subjects with respect to the Gly482Ser and +A2962G polymorphisms. Conclusions The A allele of Thr394Thr (G , A) polymorphism of the PGC-1 gene is associated with Type 2 diabetes in Asian Indian subjects and the XA genotype confers 1.6 times higher risk for Type 2 diabetes compared with the GG genotype in this population. [source]

Cytology of metastatic cervical squamous cell carcinoma in pleural fluid: Report of a case confirmed by human papillomavirus typing

DIAGNOSTIC CYTOPATHOLOGY, Issue 5 2009
Roberto G. Gamez M.D.
Abstract Cervical squamous cell carcinomas are rarely the cause of malignant effusions. Their identification can be relatively easy when keratinizing atypical squamous cells are present, but may be very difficult when only nonkeratinizing malignant cells are present. We present the case of a 47-year-old woman who presented with a large left pleural effusion after having recently completed chemoradiation therapy for stage IIB cervical squamous cell carcinoma. Cytologic examination of the fluid showed a uniform population of single atypical cells with finely vacuolated cytoplasm, ectoendoplasmic demarcation, cell-in-cell arrangements, and short rows of cells with intervening "windows," all features reminiscent of mesothelial cells. No keratinization or three-dimensional cell clusters were identified. A panel of immunohistochemical stains was performed on the cell block material, and the atypical cells were positive for cytokeratin 5/6, p63, and p16 but not for cytokeratin 7, calretinin, WT1, or Ber-EP4 or TTF1. These findings were consistent with metastatic squamous cell carcinoma. HPV DNA determination and typing by PCR confirmed the presence of HPV16 in an aliquot of pleural fluid. This is to our knowledge the first reported case of pleural fluid involved by metastatic squamous cell carcinoma where HPV DNA testing was used to confirm the origin of the metastasis. Despite its rarity, metastatic nonkeratinizing squamous cell carcinoma should be considered when a single cell population of large atypical cells is found in effusions. Immunoperoxidase stains and HPV testing can be performed to establish the diagnosis and confirm the origin from a cervical primary. Diagn. Cytopathol. 2009. © 2009 Wiley-Liss, Inc. [source]

Are adjunctive markers useful in routine cervical cancer screening?

DIAGNOSTIC CYTOPATHOLOGY, Issue 7 2008
Application of p16INK4a, HPV-PCR on ThinPrep samples with histological follow-up
Abstract The objectives of the study were to evaluate 1) the diagnostic sensitivity and specificity of p16INK4a as a marker for high-grade cervical lesions, 2) the results of a real-time polymerase chain reaction detecting high-risk human papillomavirus, and 3) the interobserver variability of the p16INK4a interpretation. A total of 232 ThinPrep samples were stained for p16INK4a, and HPV-DNA PCR was performed on 107 specimens with inclusion of both benign and abnormal cytology. Histological follow-up information was collected. The diagnostic sensitivity of ASC+ with CIN2+ in histology as endpoint was 96% for p16INK4a and 100% for HR-HPV DNA PCR, and the diagnostic specificity was 41% and 27%, respectively. If p16INK4a had been used for triage of the ASC samples, then 18 patients (42%) could have been spared unnecessary follow-up procedures compared to six patients (21%) with the HR-HPV DNA test. Diagn. Cytopathol. 2008;36:453,459. © 2008 Wiley-Liss, Inc. [source]

Human T-lymphotropic virus type-1 related adult T-cell leukemia/lymphoma presenting as a parotid mass diagnosed by fine-needle aspiration biopsy

DIAGNOSTIC CYTOPATHOLOGY, Issue 5 2004
Guo-Xia Tong M.D., Ph.D.
Abstract A 48-yr-old black woman with a history of blood transfusions for menorrhagia secondary to uterine fibroids but no known Caribbean association presented with a 6-wk history of a rapidly enlarging right parotid mass. At the time of presentation, she could not close her right eye. An aspiration biopsy showed small, medium, and large lymphoma cells with angulated nuclei, red macronucleoli, and basophilic cytoplasm with fine vacuoles. Flow cytometry indicated a (CD25+/CD7,) T-cell lineage, suggesting an human T-lymphotropic virus (HTLV) 1-related T-cell leukemia/lymphoma, which was confirmed by polymerase chain reaction (PCR)-based amplification on DNA extracted from fresh tissue with specific oligonucleotide primers for HTLV-1 DNA sequence. Histology showed interstitial infiltration and destruction of the parotid parenchyma by lymphoma cells without involvement of adjacent lymph nodes. Total body CT scan and magnetic resonance imaging (MRI) studies were negative for lymphadenopathy but showed liver metastasis. To our knowledge, this is the first reported case of HTLV-1-related primary parotid lymphoma as the initial presentation of adult T-cell leukemia/lymphoma. Diagn. Cytopathol. 2004;31:333,337. © 2004 Wiley-Liss, Inc. [source]