PubMed Health⌕ Search

Biomedical subjects

M Lab

Publications and source records attributed to M Lab.

At least 19 recordsLinked to original sources
PubMed2006

Genes encoding bile acid, phospholipid and anion transporters are expressed in a human fetal cardiomyocyte culture.

OBJECTIVES: To establish a human fetal cardiomyocyte culture and to investigate whether the genes that encode transporters that may influence influx or efflux of bile acids are expressed in human fetal cardiomyocytes. DESIGN: Laboratory study. SETTING: Imperial College London. SAMPLE: Six fetal hearts were obtained at the time of termination of pregnancy at 12-13 weeks of gestation and used to generate primary human cardiomyocyte cultures. METHODS: To confirm the presence of cardiomyocytes, the cells were incubated with monoclonal antibodies to sarcomeric alpha-actinin and anticardiac myosin heavy chain. Real-time reverse transcription polymerase chain reaction was used to establish whether transcripts of genes that may influence bile acid transport are present in the culture (NTCP, BSEP, MDR3, FIC1, MRP2, MRP3, OATP-A, OATP-C, OATP-D, OATP-E) and whether taurocholate administration alters messenger RNA (mRNA) expression. MAIN OUTCOME MEASURES: Relative mRNA expression of genes of interest. RESULTS: Real-time polymerase chain reaction demonstrated the presence of mRNA for BSEP, MDR3, FIC1, OATP-C, OATP-D and OATP-E in fetal heart. Four transcripts remained in the cardiomyocyte culture (BSEP, MDR3, FIC1 and OATP-D), and we demonstrated the influence of taurocholate on gene expression. CONCLUSIONS: We have developed an in vitro model of the fetal heart that may be used for studies of the cardiac effect of endobiotics, e.g. bile acids, or of specific agents that may be used to treat the mother or fetus in pregnancy.

Bile Acids and Salts↗
PubMed2004

Comparison of the arrhythmogenic effects of tauro- and glycoconjugates of cholic acid in an in vitro study of rat cardiomyocytes.

Obstetric cholestasis is associated with intrauterine death. In obstetric cholestasis, primary bile acids are more commonly conjugated with taurine than glycine, while glycoconjugates predominate in normal pregnancy. Using an in vitro model of rat cardiomyocytes, we compared the effect of tauro- and glycoconjugated cholate on cardiomyocyte rhythm, contraction amplitude and network integrity. We demonstrated that taurocholate had a more marked effect on all of these parameters, and the effects of the glycoconjugates were fully reversible while those of tauroconjugates were not. The increased proportion of tauroconjugated bile acids in obstetric cholestasis may contribute to the aetiology of the intrauterine death associated with the condition.

Animals↗
PubMed2003

Dexamethasone and ursodeoxycholic acid protect against the arrhythmogenic effect of taurocholate in an in vitro study of rat cardiomyocytes.

OBJECTIVE: To establish whether the therapeutic agents ursodeoxycholic acid and dexamethasone protect cardiomyocytes from taurocholate-induced arrhythmias in an in vitro model. DESIGN: Laboratory study. SETTING: Imperial College London, Hammersmith Campus. SAMPLE: Neonatal rat cardiomyocytes. METHODS: Using scanning ion conductance microscopy, we measured the rate, rhythm, amplitude of contraction and calcium dynamics of ventricular myocytes from one to two day old rats. Cells were pre-incubated for 16 hours in dexamethasone (80 or 800 nM) or 0.1 mM ursodeoxycholic acid before adding taurocholate at different concentrations (0.3-4.5 mM). MAIN OUTCOME MEASURES: Changes in rate and amplitude of contraction, calcium dynamics and rhythm. RESULTS: Taurocholate at concentrations of up to 3 mM induces abnormal changes including reductions in rate, amplitude of contraction, abnormal calcium dynamics and dysrhythmias. Although dexamethasone had no immediate protective effect on these changes, pre-incubation with dexamethasone was protective. Ursodeoxycholic acid pre-incubation was protective at taurocholate concentrations up to 1 mM. CONCLUSION: The therapeutic agents dexamethasone and ursodeoxycholic acid appear protective against the arrhythmogenic effect of taurocholate on cardiomyocytes.

Animals↗
PubMed2001

Heterogeneous changes of monophasic action potential induced by sustained stretch in atrium.

INTRODUCTION: Chronic enlargement of atrium is common in atrial fibrillation, and the effects of stretch on atrial action potentials seem inconsistent. As atrial muscle is heterogeneous, we suggest that atrial stretch induces a variable electrophysiologic response and that the effects of stretch are only partially mediated by stretch-activated channels. METHODS AND RESULTS: Sixteen guinea pig hearts were perfused by the Langendorff method using Kreb's solution with and without 80 microM streptomycin, which is a stretch-activated channel blocker. Suction electrodes were used to record monophasic action potentials (MAPs) of the left atrium. Left auricular pressure was monitored by a balloon. We determined the MAP duration at 50% and 90% repolarization (MAPD50 and MAPD90) in basal conditions and after a slow onset but sustained stretch of the atrium in the absence and presence of streptomycin. Stretch induced no overall consistent or significant change in mean MAPD50 and MAPD90. The individual responses were markedly variable. The most frequent response (about 50%) was a decrease in MAPD50 and MAPD90. In 25% of cases, there was no change, and in 25% we observed increases in MAPD50 and MAPD90. Streptomycin did not affect MAPD50 and MAPD90, but it dramatically modified the distribution of MAPD changes induced by stretch. In particular, streptomycin removed stretch-induced shortening of MAPD50 and MAPD90, whereas it did not affect stretch-induced lengthening. CONCLUSION: Sustained stretch of atrium induces variable modifications of MAPD that are only partially inhibited by streptomycin. This suggests the participation of ionic channels other than specific stretch-activated channels in the response of atrial myocardium to sustained stretch.

Action Potentials↗
PubMed2001

The bile acid taurocholate impairs rat cardiomyocyte function: a proposed mechanism for intra-uterine fetal death in obstetric cholestasis.

Obstetric cholestasis is a liver disease of pregnancy that can be complicated by sudden, hitherto unexplained, intra-uterine fetal death. Because intra-uterine death occurs suddenly, and because fetal heart rate abnormalities have been reported in obstetric cholestasis, we hypothesized that intra-uterine death is caused by impaired fetal cardiomyocyte function, resulting in fetal cardiac arrest. Obstetric cholestasis is associated with raised levels of maternal and fetal serum bile acids, and we propose that these may alter cardiomyocyte function. It was not possible to investigate the effects of bile acids on the intact human fetal heart at a cellular level. Therefore we used the closest available model of fetal myocardium at term: a primary culture of neonatal rat cardiomyocytes in which cells beat synchronously and develop pacemaker activity. The effect of the primary bile acid taurocholate (0.3 mM and 3 mM) on cultures of single cardiomyocytes, each with its own independent rate of contraction, was a reversible decrease in the rate of contraction and in the proportion of beating cells (P < 0.001). Addition of taurocholate to a network of synchronously beating cells caused a similar decrease in the rate of contraction. Furthermore, the integrity of the network was destroyed, and cells ceased to beat synchronously. Taurocholate also resulted in altered calcium dynamics and loss of synchronous beating. These data suggest that raised levels of the bile acid taurocholate in the fetal serum in obstetric cholestasis may result in the development of a fetal dysrhythmia and in sudden intra-uterine death.

Animals↗
PubMed1999

Direct in situ reverse transcriptase-linked polymerase chain reaction with biotinylated primers for the detection of hepatitis C virus RNA in liver biopsies.

To assess the presence and the cellular distribution of hepatitis C virus (HCV) RNA in the liver of 11 patients with confirmed HCV infection, a direct in situ reverse transcriptase-linked polymerase chain reaction (RT-PCR) method was performed on formalin-fixed and paraffin-embedded biopsies. The oligonucleotide primers used were specific to the 5' non coding region. An unlabelled downstream oligonucleotide served as a primer for reverse transcription as well as PCR. The upstream oligonucleotide serving as a primer for PCR was biotinylated, allowing a direct enzymatic detection of PCR products. HCV infected cells revealed cytoplasmic staining mainly concentrated towards the interface of the nucleus and cytoplasm. Most of the stained cells were hepatocytes and sometimes Kupffer cells. The results were compared with those obtained by RT-PCR of RNA extracted from the corresponding tissue block. Extracted HCV RNA could be detected in liver tissues of nine out of 11 (82%) infected patients. The detection rate using in situ RT-PCR was 7/11 (63%). The use of labelled primers improved specificity of direct in situ methods, by preventing non-specific incorporation of labelled dNTPs into fragmented DNA. Further studies are however required in order to increase detection sensitivity of HCV infection by in situ molecular methods.

Biotin↗
PubMed1999

Human papillomavirus detection by non isotopic in situ hybridization, in situ hybridization with signal amplification and in situ polymerase chain reaction.

Classical in situ hybridization (ISH) with biotinylated probes makes it possible to detect and localize human papillomavirus (HPV) nucleic acid sequences in cytological and histological materials. This method is however of limited value in the detection of a few copies of the virus. Moreover the specificity of such a technique is not always convincing when ISH signals are small and/or of low intensity. Recently, much attention has been focused on the utility of the in vitro polymerase chain reaction (PCR) and especially on PCR-single strand conformation polymorphism (SSCP) to amplify small amounts of viral DNA with accurate hybrid specificity. But the latter method requires nucleic acid extraction and tissue destruction. Thus, correlation between the PCR results and histological findings is not possible. Hence, the aim of our current study was to apply to HeLa cells and cervical formalin-fixed and paraffin-embedded biopsies, a novel procedure of ISH signal amplification, the catalyzed signal amplification (CSA). Such a procedure is based on the deposition of streptavidin-horseradish peroxidase catalyzing the deposition of biotinylated tyramide molecules on the location of the probed target. The biotin accumulation is then detected with streptavidin peroxidase and diaminobenzidine. The results were compared with those obtained by direct and indirect in situ PCR. The catalysed signal amplification successfully increased the sensitivity and efficiency of ISH for the detection of rare sequences in HPV infected cells and histological materials. Such a method was found simpler and faster than in situ PCR and tissue morphology was better preserved.

Biotin↗
PubMed1998

Molecular and functional characterization of a recombinant protein of Trichuris trichiura.

The pore-forming protein of the human whipworm, Trichuris trichiura, has been postulated to facilitate invasion of the host gut and enable the parasite to maintain its syncytial environment. The data presented here describe the first, to our knowledge, molecular characterization of a pore-forming protein in any helminth and provide a unique demonstration of the functional interaction between a parasite antigen and host molecules. Immunological screening of a T. trichiura cDNA library with T. trichiura infection sera identified a clone of 1.4 kB, the cDNA consisting of 1495 base pairs encoding a protein of 50 kDa. The sequence has a highly repetitive nature containing nine four-disulphide-bonded core domains. Structural prediction analyses reveals an amphipathic nature. TT50 induced pore formation in bilayers in a manner identical to that of the native protein. IgG antibody isolated from T. trichiura infection serum was observed to abolish channel activity.

Amino Acid Sequence↗
PubMed1997

[Obliterative arteriopathy in chronic rejection after hepatic transplantation. Search of cytomegalovirus genome by in situ hybridization and viral antigens by immunohistochemistry].

OBJECTIVES: This study was performed to assess the role of cytomegalovirus in the parietal infection of intrahepatic arteries in the pathogenesis of obliterative arteriopathy from chronic rejection after orthotopic liver transplantation. METHODS: We studied two groups of liver transplants by in situ hybridization and immunohistochemistry: group 1, including 10 liver grafts with obliterating arteriopathy, and group 2 including 7 liver grafts without any arterial disorders. The results were correlated with clinical data (cytomegalovirus infection and acute rejection). RESULTS: By in situ hybridization, cytomegalovirus DNA was identified in the media in 70% of transplants in group 1 and 42% in group 2. Detection of immediate early and late antigens by immunohistochemistry was negative. Cytomegalovirus infections were often associated with acute rejection. CONCLUSION: These results suggest that cytomegalovirus detected in arteries is latent, and that cytomegalovirus probably does not play a role in the pathogenesis of chronic rejection obliterative arteriopathy.

Adult↗
PubMed1996

Human ventricular action potential duration during short and long cycles. Rapid modulation by ischemia.

BACKGROUND: Mechanisms underlying the initiation of ventricular arrhythmias in ischemia by a premature beat or after a pause remain unclear. The kinetics of electrical restitution, which is the modulation of action potential duration (APD) by an abrupt alteration in cycle length, may be important. METHODS AND RESULTS: We recorded one or two simultaneous monophasic action potentials (MAPs) from the right ventricular septum during balloon occlusion of the left anterior descending coronary artery (LAD) (14 patients), which is expected to induce ischemia at the recording site, and during occlusion of the right coronary artery (RCA) (7 patients), which is not expected to induce ischemia at the recording area. The latter acted as a control. A test pulse sequence was incorporated whereby during steady-state pacing, test beats of altered cycle length were interpose. During LAD occlusion, APD for basic beats shortened from 260 +/- 4 to 236 +/- 4 ms (P < .0001), whereas the control group (RCA occlusion) showed no significant change (251 +/- 7 to 249 +/- 9 ms; P = NS). LAD occlusion resulted in flattening of the slope relating APD of test beats to diastolic interval (P = .001), whereas in the control group (RCA occlusion) the slope remained unchanged. Similar results were obtained during a second occlusion. CONCLUSIONS: LAD occlusion in patients during balloon angioplasty shortened MAP duration of basic beats and minimized, abolished, or reversed the normal APD/diastolic-interval relation of test beats of altered cycle length at sites served by the occluded vessel. The results suggest that ischemia flattens the electrical restitution curve in the human endocardium. These findings may have important implications in arrhythmogenesis.

Action Potentials↗
PubMed1995

Detection of human cytomegalovirus in female and male anogenital lesions with papillomavirus-associated dysplastic changes.

The aim of the study was to determine the presence of human papillomavirus (HPV) and cytomegalovirus (CMV) DNA in lesions from mucosal and keratinized cutaneous epithelium of the anogenital tract. One hundred ninety-eight women and 55 men with anogenital lesions were enrolled for the study. Biopsies were prepared for histological analysis and in situ hybridization using biotinylated probes. Potential oncogenic HPV were found in 15% of samples showing no outward signs of HPV infection or inflammation. They were detected in 40% of samples showing low grade squamous intra-epithelial lesions (SIL) and in 95 to 100% of lesions showing high-grade SIL. Cytomegalovirus DNA was detected twice as frequently in women as in men, and was preferentially associated with high-grade SIL. A significant relationship between high-risk HPVs and the presence of CMV was observed in the low-grade lesions. Our data suggest that the CMV genome in the host epithelial cells might act as a permissive factor upon which oncogenic HPV could interact over time to increase the frequency and severity of cervical, penile, and anal dysplasia.

Anus Neoplasms↗
PubMed1995

[Detection of cyclin A mRNA in intra-epithelial lesions of the anogenital tract induced by papillomavirus].

Human papillomaviruses (HPVs) have been recognized as likely viral agents responsible for anogenital precancer lesions and squamous cell cancers in both men and women. Nevertheless their role in carcinogenesis is not entirely clear. There are many other agents, both viral and non-viral, which might act synergistically or separately with HPV in a multistep tumorigenic process. Among non-viral factors, protooncogene and tumor suppressor gene alterations may be a major step towards the malignant transformation of an HPV-infected cell. The induction of unscheduled DNA synthesis and cell proliferation by human papillomaviruses would provide the basis for the potential of these viruses to contribute to the formation of tumors in vivo. Thus, we studied by in situ hybridization (ISH) the cyclin A gene expression in HPV-induced condylomatous and dysplastic lesions of the anogenital tract, and in inflammatory squamous intraepithelial tissues. We observed a high level of cyclin A gene expression in low grade squamous intraepithelial lesions infected by HPV type 6/11. Cyclin A induction was surprisingly more important in the upper third layers of differentiated cells together with large amounts of HPV DNA, than in proliferating basal and parabasal cells. An identical pattern was also shown in some low grade squamous intraepithelial lesions infected with potential oncogenic HPV. In contrast, there was evidence of low abundance cyclin A mRNA in most high grade squamous intraepithelial lesions induced by high risk HPV. In the inflammatory tissues, an ISH signal was sometimes detected in the basal cells. As for proliferating cell nuclear antigen (PCNA), these results observed in vivo reveal that viral oncoproteins are able to reactivate cellular DNA replication machinery to support papillomavirus DNA replication in normally differentiated, non-cycling cells. The induction of cyclin A gene expression appears to correlate with the proliferative rather than the transforming properties of these cells.

Anus Diseases↗
PubMed1994

Detection of human papillomavirus by in situ polymerase chain reaction in paraffin-embedded cervical biopsies.

The efficiency of the in situ polymerase chain reaction (PCR) for the detection of human papillomavirus (HPV) DNA sequences in 20 cervical biopsies fixed with buffered formalin, paraffin-embedded and revealed negatively by conventional in situ hybridization (ISH) has been investigated. The biopsies were classified histologically into condylomata acuminata without dysplasia, cervical intraepithelial neoplasia and carcinoma in situ. Amplified HPV DNA was performed after an optimal proteolytic digestion using one pair of consensus oligonucleotide primers located in the L1 ORF of HPV 6 and ISH was carried out after the PCR assay with a cocktail of biotinylated HPV probes. Viral DNA was detected in 100% of high grade squamous intraepithelial lesions (SIL) and in 50 to 60% of low grade SIL. The high sensitivity of the in situ PCR applicable to paraffin-embedded archival biopsies facilitated the detection of cells poorly reactive by conventional ISH. In situ PCR appeared clearly an efficient tool to investigate HPV infection in tissue sections.

Base Sequence↗
PubMed1994

Rapid detection of active cytomegalovirus infection by in situ polymerase chain reaction on MRC5 cells inoculated with blood specimens.

An in situ polymerase chain reaction was developed to amplify immediate early genes of human cytomegalovirus in cells cultured in a 96 well plate and infected with leukocytes. The technical parameters enabling optimal detection of the DNA sequences were defined. The key to this method is the fixation of cells, which facilitates the access of the PCR mixture into the cell nuclei and preserves cell morphology. Such a technique could have wide application for the detection and identification of other infectious viruses in cultured cells very early after inoculation of clinical samples.

Base Sequence↗
PubMed1994

Evaluation of Biohit HPV Screening and Typing kits in detection of human papillomavirus DNA from lesions of anogenital tract.

The Biohit HPV Screening and Typing kits for in situ hybridization of human papillomavirus (HPV) DNA are now commercially available. The HPV Screening kit contains a cocktail of HPV probes, and the Typing kit contains separated hybridization probes for HPV 6, 11, 16, 18, 31, and 33. They were evaluated by comparison with an in situ hybridization (ISH) method, using the Pathogene HPV probes 6/11, 16/18, 31/33/51. One hundred anogenital biopsies from 78 women and 22 men were tested. Among them, 43% showed normal or inflammatory mucosa, 44%, koilocytosis or mild dysplasia, and 13%, moderate to severe dysplasia. Altogether, 60 specimens were positive with the ISH reference method: 17 with the HPV 6/11 probe, 12 with the HPV 16/18 probe, 16 with the HPV 31/33/51 probe, and 15 had mixed infections. The agreement between the Screening test and the homemade ISH is 91%. The Screening test has a sensitivity of 93% and a specificity of 87%. As for the Biohit Typing test, four false-negative samples, and partial or total discordance in nine and four samples, respectively, were observed when compared to our reference method. Thus the agreement between both typing ISH tests is 92%. The sensitivity of the Biohit Typing test is 93%, and the specificity, 91%. The sensitivity decreases to 72% when the 31 and 33 probes are evaluated separately. The Biohit Screening assay is simple, reliable, reproducible, and suitable for rapid routine screening. The Biohit Typing test allows the detection of a specific type of HPV DNA and also permits, in mixed HPV infection, definition of the type of associated HPV DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Anus Diseases↗