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PubMed2002

Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes).

Novel fluorescent oligonucleotides that contain a 3' minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5' nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. Common spreadsheet software was used for automated genotype assignment. As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Hum Mutat 19:554-559, 2002.

Alleles↗
PubMed1996

Minimal residual disease post-bone marrow transplantation for hemato-oncological diseases.

The detection of minimal residual disease (MRD), which is important in cancer treatment, gained special significance in bone marrow transplantation (BMT-) due to the possibility not just to detect but recently also to prevent, treat and reinduce remission in patients that relapsed post-BMT by immunotherapy. The various modern techniques of MRD detection are described including cytogenetics, analysis of restriction fragment length polymorphism, variable number of tandem repeats by Southern Blot or polymerase chain reaction (PCR), microsatellite sequences, PCR amplification products of the Y chromosome or the Amelogenin gene, quantitative PCR and fluorescence in situ hybridization. The role of MRD detection in refinement of indications for BMT, autografting, prediction of relapse, adoptive immunotherapy, mixed chimerism in nonmalignant diseases and in solid organ transplantation is discussed.

Bone Marrow Transplantation↗
PubMedDate not supplied

Re-emergence of Chlamydia trachomatis infection after mass antibiotic treatment of a trachoma-endemic Gambian community: a longitudinal study.

BACKGROUND: Community-wide mass antibiotic treatment is a central component of trachoma control. The optimum frequency and duration of treatment are unknown. We measured the effect of mass treatment on the conjunctival burden of Chlamydia trachomatis in a Gambian community with low to medium trachoma prevalence and investigated the rate, route, and determinants of re-emergent infection. METHODS: 14 trachoma-endemic villages in rural Gambia were examined and conjunctival swabs obtained at baseline, 2, 6, 12, and 17 months. Mass antibiotic treatment with azithromycin was given to the community at baseline. C trachomatis was detected by qualitative PCR and individual infection load then estimated by real-time quantitative PCR. FINDINGS: C trachomatis was detected in 95 (7%) of 1319 individuals at baseline. Treatment coverage was 83% of the population (1328 of 1595 people). The effect of mass treatment was heterogeneous. In 12 villages all baseline infections (34 [3%] of 1062 individuals) resolved, and prevalence (three [0.3%]) and infection load remained low throughout the study. Two villages (baseline infection: 61 [24%] of 257 individuals) had increased infection 2 months after treatment (74 [30%]), after extensive contact with other untreated communities. Subsequently, this value reduced to less than half of that before treatment (25 [11%]). INTERPRETATION: Mass antibiotic treatment generally results in effective, longlasting control of C trachomatis in this environment. For low prevalence regions, one treatment episode might be sufficient. Infection can be reintroduced through contact with untreated populations. Communities need to be monitored for treatment failure and control measures implemented over wide geographical areas.

Administration, Oral↗
PubMed2003

Presence and significance of TT virus in Danish patients on maintenance hemodialysis.

OBJECTIVES: To determine the prevalence of TT virus (TTV) in a population of Danish hemodialysis patients and evaluate possible relations between TTV infection and elevated levels of C-reactive protein (CRP) and hypo-response to treatment with erythropoietin (EPO). MATERIAL AND METHODS: Patients on maintenance hemodialysis at a single center were invited to participate. Demographic and clinical data were registered. Blood samples for virological and routine biochemical tests were drawn simultaneously. TTV DNA was detected using polymerase chain reaction (PCR). TTV viral load was estimated by means of semi-quantitative PCR. All patients were tested for hepatitis B, hepatitis C and GB virus C. RESULTS: Of 252 patients, 204 (80.9%) gave their written informed consent to participate in the study. The prevalence of TTV was 68% and 50% of TTV-positive patients had a high TTV viral load. TTV-positive patients were significantly older than TTV-negative patients (p = 0.011). No relations were found between TTV infection and elevated levels of alanine aminotransferase (ALT) or CRP or hypo-response to EPO treatment. The mean hemoglobin concentration was 11.24 +/- 1.48 g/dl. Patients with a high TTV viral load had a lower level of hemoglobin (10.86 +/- 1.47 g/dl) than the others (p = 0.01). This trend suggested a positive relation between TTV infection and the number of blood transfusions. A restriction fragment length polymorphism assay suggested that patients were infected with different TTV strains. CONCLUSIONS: TTV is common in patients on maintenance hemodialysis. The presence of TTV is associated with increasing age. Patients with a high TTV viral load had lower levels of hemoglobin than the others. TTV infection is not related to elevated levels of ALT or CRP or to hypo-response to EPO treatment.

Adolescent↗
PubMed2001

Feline leukaemia provirus load during the course of experimental infection and in naturally infected cats.

Feline leukaemia virus (FeLV) infection in domestic cats can vary in its outcome (persistent, transient, no infection) for reasons that are not entirely known. It was hypothesized that the initial virus and provirus load could significantly influence the course of retrovirus infection. To determine the role of provirus loads, two methods of PCR, a nested PCR and a fluorogenic probe-based (TaqMan) real-time quantitative PCR, which were specific to the U3 region of FeLV-A were established. FeLV provirus in naturally and experimentally infected cats was then measured. Only 3 weeks after experimental FeLV-A infection, persistently infected cats demonstrated higher provirus loads and lower humoral immune responses than cats that had overcome antigenaemia. Lower initial provirus loads were associated with successful humoral immune responses. Unexpectedly, provirus in the buffy-coat cells of two cats that tested negative for the p27 antigen (a marker for viraemia) was also detected. In 597 Swiss cats, comparison of p27 antigen levels with PCR results revealed broad agreement. However, similar to the experimental situation, a significant number of animals (10%) was negative for the p27 antigen and FeLV-positive by PCR. These cats had a mean provirus load 300-fold lower than that of animals testing positive for the p27 antigen. In conclusion, an association between the provirus load and the outcome of FeLV infection was found. Detection of provirus carriers should contribute to further the control of FeLV. In addition, quantification of provirus loads will lead to a better understanding of FeLV pathogenesis and anti-retrovirus protective mechanisms.

Animals↗
PubMed1999

Activity of the c-myc replicator at an ectopic chromosomal location.

DNA replication starts at multiple discrete sites across the human chromosomal c-myc region, including two or more sites within 2.4 kb upstream of the c-myc gene. The corresponding 2.4-kb c-myc origin fragment confers autonomously replicating sequence (ARS) activity on plasmids, which specifically initiate replication in the origin fragment in vitro and in vivo. To test whether the region that displays plasmid replicator activity also acts as a chromosomal replicator, HeLa cell sublines that each contain a single copy of the Saccharomyces cerevisiae FLP recombinase target (FRT) sequence flanked by selectable markers were constructed. A clonal line containing a single unrearranged copy of the transduced c-myc origin was produced by cotransfecting a donor plasmid containing the 2.4-kb c-myc origin fragment and FRT, along with a plasmid expressing the yeast FLP recombinase, into cells containing a chromosomal FRT acceptor site. The amount of short nascent DNA strands at the chromosomal acceptor site was quantitated before and after targeted integration of the origin fragment. Competitive PCR quantitation showed that the c-myc origin construct substantially increased the amount of nascent DNA relative to that at the unoccupied acceptor site and to that after the insertion of non-myc DNA. The abundance of nascent strands was greatest close to the c-myc insert of the integrated donor plasmid, and significant increases in nascent strand abundance were observed at sites flanking the insertion. These results provide biochemical and genetic evidence for the existence of chromosomal replicators in metazoan cells and are consistent with the presence of chromosomal replicator activity in the 2.4-kb region of c-myc origin DNA.

Chromosomes, Human↗
PubMed2004

Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and host specific colonisation during Helicobacter pylori infection.

BACKGROUND AND AIMS: Recent studies linked cytokine gene polymorphisms to H pylori related gastric cancer development. The current study evaluated the role of cytokine gene polymorphisms for mucosal cytokine expression, the gastric inflammatory response, and bacterial colonisation during H pylori infection. PATIENTS AND METHODS: In 207 H pylori infected patients with chronic gastritis, polymorphisms at different loci of the interleukin (IL)-10, IL-1B, IL-1 receptor antagonist (IL-1RN), tumour necrosis factor (TNF)-A, and interferon (IFN)-G genes were genotyped by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis, and allelic discriminating TaqMan PCR. Mucosal cytokine mRNA copy numbers were determined by real time quantitative PCR. Presence of bacterial virulence factors was investigated by cagA, vacAs1/2, and babA2 PCR. Biopsies were assessed with regard to the degrees of granulocytic/lymphocytic infiltration and the presence of intestinal metaplasia (IM) and atrophic gastritis (AG). RESULTS: Proinflammatory IL-1 polymorphisms (IL-1RN*2(+)/IL-1B-511T/-31C(+)) were associated with increased IL-1beta expression, more severe degrees of inflammation, and an increased prevalence of IM and AG. Carriers of the IL-10-1082G/-819C/-592C alleles (GCC haplotype) had higher mucosal IL-10 mRNA levels than ATA haplotype carriers and were associated with colonisation by more virulent cagA(+), vacAs1(+), and babA2(+) H pylori strains. The TNF-A-307(G/A) and IFN-G+874(A/T) polymorphisms did not influence mucosal cytokine expression or the inflammatory response to H pylori. CONCLUSIONS: Cytokine gene polymorphisms influence mucosal cytokine expression, gastric inflammation, and the long term development of precancerous lesions in H pylori infection. Host polymorphisms are associated with certain bacterial strain types, suggesting host specific colonisation or adaptation. These findings contribute to the understanding of the complex interplay between host and bacterial factors involved in the development of gastric pathology.

Adult↗
PubMed2004

Quantification of ribozyme target RNA using real-time PCR.

An important part of the ribozyme efficiency-screening process is to have a fast and accurate way to measure steady-state levels of the target RNA. Here, we describe the use of real-time polymerase chain reaction (PCR) for quantification of ribozyme target transcripts. In contrast to classical quantitative PCR, real-time PCR does not require extensive manipulation or generation of relatively complex reagents, thus reducing the risk of contamination. PCR products generated by Taq polymerase in the presence of SYBR Green dye I can be monitored each cycle by collecting fluorescence signals emitted only as the double-stranded DNA is formed. The temperature at which the fluorescent data used for quantification are collected is based on the melting-curve analysis of the amplified product. After constructing a standard curve by plotting the log of the standards' copy number vs their fractional cycle number, the copy number of the unknown samples is automatically determined by interpolation of this curve. However it is very important to validate the melting curve profile with standard gel electrophoresis, particularly while setting up the technique. Real-time PCR is fast and reproducible. Excluding the isolation of RNA and synthesis of cDNA, the results can be obtained in less than 1 h. The coefficient of variance is 15% in the range of 104-106 gene copies.

Base Sequence↗
PubMed2004

Temporal analysis of Coxiella burnetii morphological differentiation.

Coxiella burnetii undergoes a poorly defined developmental cycle that generates morphologically distinct small-cell variants (SCV) and large-cell variants (LCV). We developed a model to study C. burnetii morphogenesis that uses Vero cells synchronously infected with homogeneous SCV (Nine Mile strain in phase II) harvested from aged infected cell cultures. A time course transmission electron microscopic analysis over 8 days of intracellular growth was evaluated in conjunction with one-step growth curves to correlate morphological differentiations with growth cycle phase. Lag phase occurred during the first 2 days postinfection (p.i.) and was primarily composed of SCV-to-LCV morphogenesis. LCV forms predominated over the next 4 days, during which exponential growth was observed. Calculated generation times during exponential phase were 10.2 h (by quantitative PCR assay) and 11.7 h (by replating fluorescent focus-forming unit assay). Stationary phase began at approximately 6 days p.i. and coincided with the reappearance of SCV, which increased in number at 8 days p.i. Quantitative reverse transcriptase-PCR demonstrated maximal expression of scvA, which encodes an SCV-specific protein, at 8 days p.i., while immunogold transmission electron microscopy revealed degradation of ScvA throughout lag and exponential phases, with increased expression observed at the onset of stationary phase. Collectively, these results indicate that the overall growth cycle of C. burnetii is characteristic of a closed bacterial system and that the replicative form of the organism is the LCV. The experimental model described in this report will allow a global transcriptome and proteome analysis of C. burnetii developmental forms.

Animals↗
PubMed2006

Quantitative measurement of streptomycetes using real-time PCR.

A quantitative real-time PCR method was developed and used for determination of streptomycetes in indoor dust samples of five homes collected during three years. The specificity of the method was tested with 14 Streptomyces and ten non-streptomycetous species, revealing a high specificity for mesophilic streptomycetes. Thermophilic species and S. albus were not efficiently detected. The method gave reproducible results in replicate analyses of the same dust DNA as well as of duplicate DNA isolations. The amount of streptomycetes in house dust was lowest in winter, followed by summer, and highest in spring and fall. The greatest variation in Streptomyces-concentrations was observed in the spring and fall samples.

Air Pollution, Indoor↗
PubMed2005

Comparison of immunocytochemistry, real-time quantitative RT-PCR and flow cytometry for detection of minimal residual disease in neuroblastoma.

A quantitative and precise measure of treatment response is warranted in neuroblastoma patients. We compared three quantitative methods often used for detection of minimal residual disease in such patients. Specificity, sensitivity and concordance of immunocytochemistry, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry were compared using experimental cell suspensions (n = 8) and clinical samples (n = 126). Neuroblastoma cells were identified by immunocytochemistry and flow cytometry using anti-GD2 (14.G2a) and anti-NCAM (5.1H11) antibodies, whereas tyrosine hydroxylase mRNA was the molecular target for quantitative RT-PCR. The sensitivity using flow cytometry was 1-2 logs less than using immunocytochemistry or quantitative RT-PCR. All control samples (n = 35) tested negative by immunocytochemistry, whereas 2/34 (6%) and 1/14 (7%) were false positive by quantitative RT-PCR and flow cytometry respectively. Concordant results were obtained in 85% of patient samples (n = 116) analyzed in parallel by quantitative RT-PCR and immunocytochemistry, whereas 71% of samples analyzed by flow cytometry and immunocytochemistry were concordant (n = 35). The correlation between tumor cell levels analyzed by quantitative RT-PCR and immunocytochemistry was high (r = 0.78, p < 0.001). Quantitative RT-PCR and immunocytochemistry both reliably detected very low levels of neuroblastoma cells in clinical samples. The agreement and correlation between these methods were high. In comparison, flow cytometry was less sensitive.

Antibodies↗
PubMed2000

Identification and characterization of a 19q12 amplicon in esophageal adenocarcinomas reveals cyclin E as the best candidate gene for this amplicon.

Genomic DNA amplification in tumors is frequently associated with an increased gene copy number of oncogenes or other cancer-related genes. We have used a two-dimensional whole-genome scanning technique to identify gene amplification events in esophageal adenocarcinomas. A multicopy genomic fragment from a tumor two-dimensional gel was cloned, and genomic amplification encompassing this fragment was confirmed by Southern blot analysis. The corresponding DNA sequence was matched by BLAST to a BAC contig, which allowed the use of electronic-PCR to localize this amplicon to 19q12. Sequence tagged site-amplification mapping, an approach recently implemented in our laboratory (Lin, L. et al., Cancer Res., 60: 1341-1347,2000), was used to characterize the amplicon. Genomic DNA from 65 esophageal and 11 gastric cardia adenocarcinomas were investigated for 19q12 amplification using quantitative PCR at 11 sequence tagged site markers neighboring the cloned fragment. The amplicon was narrowed from >8 cM to a minimal critical region spanning <0.8 cM, between D19S919 and D19S882. This region includes the cyclin E gene. Fourteen expressed sequence tags (ESTs) covering the minimal region were then assayed for potential gene overexpression using quantitative reverse transcription-PCR. Seven of the selected ESTs were found to be both amplified and overexpressed. Among these seven ESTs, cyclin E showed the highest frequency of gene amplification and overexpression in the tumors examined, which allowed us to finalize the core-amplified region to <300 kb. These results indicate that cyclin E is the likely target gene selected by the amplification event at 19q12. The fact that cyclin E overexpression was found only in the amplified tumors examined indicates that gene amplification underlies the cyclin E gene overexpression. Our study represents the first extensive analysis of the 19q12 amplicon, and is the first to physically map the core-amplified domain to a region of <300 kb that includes cyclin E. Amplification of 19q12 was found neither in the 28 esophageal squamous cancers nor in the 39 lung adenocarcinomas examined but was observed in 13.8% of esophageal and 9.1% of gastric cardia adenocarcinomas.

Adenocarcinoma↗
PubMed2003

Monitoring intracellular replication of Chlamydophila (Chlamydia) pneumoniae in cell cultures and comparing clinical samples by real-time PCR.

Strains of Chlamydophila pneumoniae may be associated with respiratory disease or atherosclerosis. Two real-time quantitative PCR assays targeting the species-specific genes Cpn0278 and ArgR were developed to compare the in vitro growth of respiratory strains AR39 and K6 with that of atherosclerotic strain A03 and to quantify C. pneumoniae in clinical samples. A third real-time PCR assay was designed to assess contamination with Mycoplasma spp. The assays targeting C. pneumoniae detected DNA concentrations corresponding to 10(4) to 10(-4) inclusion-forming units (IFU)/reaction and were highly specific. AR39 exhibited the longest lag phase and period of exponential growth; K6 augmented growth rates at higher inocula; and A03 grew at highest rates. Contamination with Mycoplasma spp. of AR39 and A03 unlikely accounted for growth differences between them. Numbers of IFU in C. pneumoniae-positive respiratory secretions varied within 4 to 5 orders of magnitude. The assays described may prove valuable for pathogenicity studies.

Base Sequence↗
PubMed2005

Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma.

Prostate carcinogenesis is influenced by genetic alterations resulting in a biochemical condition that favors cell proliferation and survival. Studies of prostate carcinoma using comparative genomic hybridization and cDNA microarray analysis indicate that numerous biochemical processes may be affected during cellular transformation and progression to an invasive phenotype. Among the consistently observed tumor-associated changes are alterations in fatty acid metabolism that influence diverse cellular activities such as signaling, energy utilization, and membrane fluidity. Increases in fatty acid synthase (FAS) levels have been shown to be one of the earliest and most frequent molecular alterations in prostate carcinogenesis. We sought to identify tumor-associated changes in the expression of genes with functional roles associated with lipid metabolism. Defined populations of normal and neoplastic prostate epithelium were acquired by laser capture microdissection and transcript levels were measured by cDNA microarray hybridization. We determined that stearoyl-CoA desaturase (SCD) transcripts were downregulated in cancer relative to normal epithelium. These results were confirmed by quantitative PCR. Further analysis by immunohistochemical evaluation of radical prostatectomy samples employed a quantitative scoring system with a range of 0-300. The median SCD expression levels were 150, 45 and 10 for normal, PIN and carcinoma samples, respectively. Statistically significant differential SCD expression between normal and cancerous epithelium was determined at the p=0.001 level, and between PIN and prostate carcinoma at the p=0.03 level. Of these cases, 92% overexpressed fatty acid synthase (FAS) in cancerous cells and 84.7% exhibited the signature of FAS overexpression and SCD loss in prostate carcinoma as compared to normal prostate epithelium. These results indicate that loss of SCD expression is a frequent event in prostate adenocarcinoma, and further supports a role for altered lipid metabolism as a factor in the process of carcinogenesis.

Blotting, Northern↗
PubMed2006

Structural and functional analysis of a salt stress inducible gene encoding voltage dependent anion channel (VDAC) from pearl millet (Pennisetum glaucum).

We have cloned and characterized a gene encoding voltage-dependent anion channel from Pennisetum glaucum (PgVDAC). PgVDAC was identified while isolating genes that were differentially up-regulated following salt stress. The genomic organization of PgVDAC clone was well conserved compared to other plant VDAC genes in terms of number of introns, their position and phasing, however, the primary amino acid sequence of voltage dependent anion channel (VDAC) proteins did not show much conservation with other plant VDACs but their secondary and tertiary structures are well conserved as predicted by in silico structural and CD spectra analyses and results show it to be a typical membrane-spanning beta-barrel leading to the formation of pore in the membrane. The heterologous expression of PgVDAC protein in yeast strain lacking the endogenous mitochondrial VDAC gene could not functionally complement it as was also previously observed for the potato VDAC. Using real-time quantitative PCR analysis it was found that transcript expression profile of PgVDAC was quantitatively and kinetically up-regulated in response to salinity, desiccation, cold and exogenous application of salicylic acid (SA); however, there was no effect of exogenous application of abscisic acid (ABA) on its expression. Constitutive over-expression of PgVDAC appears to be deleterious in transgenic rice plant; however, low level of up-regulation imparted salinity stress adaptive response. A search for a more suitable inducible transgene system is currently under way to understand PgVDAC expression levels in plant development and its role in stress adaptation.

Amino Acid Sequence↗
PubMed1999

Interleukin-8, interleukin-10, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression levels are higher in synovial tissue from patients with rheumatoid arthritis than in osteoarthritis.

The aim of this work was to determine differences in pro- and anti-inflammatory cytokine and adhesion molecule expression in synovial tissue from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Synovial tissue samples were obtained from patients with RA and OA, and from healthy individuals. The expression of mRNA of interleukin (IL)-1beta, IL-4, IL-6, IL-8, IL-10, IL-13, tumour necrosis factor-alpha (TNF-alpha) and transforming growth-factor-beta1 (TGF-beta1) was evaluated by the polymerase chain reaction (PCR). In addition, IL-8 and IL-10 transcripts were measured by quantitative PCR. The expression of IL-8 and IL-10 proteins was determined by immunoperoxidase staining. To evaluate the inflammatory stage of synovial tissue, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein expression was also determined. RA patients were found to display higher levels of adhesion molecules than patients with OA. PCR analysis showed a similar profile of cytokine transcripts between the OA and RA groups. Gene expression of IL-4 and IL-13 in synovium was undetectable. In contrast, IL-1beta, IL-6, IL-8, IL-10, TNF-alpha and TGF-beta1 transcripts were expressed by both groups. Increased levels of IL-8 and IL-10 transcripts and their proteins were observed in synovium from RA patients when compared to patients with OA and healthy controls. Thus, our data show that IL-8, IL-10, ICAM-1 and VCAM-1 expression levels are higher in synovial tissue from patients with RA than in similar tissue from patients with OA.

Adult↗
PubMed2002

Changes in activity and expression of phosphofructokinase in different rat brain regions after basal forebrain cholinergic lesion.

Selective lesion of rat basal forebrain by the cholinergic immunotoxin 192IgG-saporin was used as an animal model to address the question of whether the changes in cortical glucose metabolism observed in patients with Alzheimer's disease may be related to impaired cholinergic transmission. At different times after creating the immunolesion, the isoenzyme pattern and steady-state mRNA levels of the key glycolytic enzyme phosphofructokinase were determined in cortex, hippocampus, basal forebrain and nucleus caudatus. The loss of cholinergic input was accompanied by a persistent decrease in choline acetytransferase and acetylcholine esterase activities in the cortical target areas similar to the cholinergic malfunction seen in Alzheimer's dementia. The basal forebrain lesion induced by the immunotoxin resulted in a transient increase in phosphofructokinase activity peaking on day 7 after inducing the lesion in cortical areas. In parallel, an increased steady-state level of phosphofructokinase mRNA was determined by RT/real-time PCR and in situ hybridization. In contrast, analysis by western blotting and quantitative PCR revealed no changes in the phosphofructokinase isoenzyme pattern after immunolesion. It is concluded that common metabolic mechanisms may underlie the degenerative and repair processes in denervated rat brain and in the diseased Alzheimer's brain.

Acetylcholinesterase↗
PubMed2002

Functional and molecular identification of ERG channels in murine portal vein myocytes.

Ion channels encoded by ether-à-go-go-related genes (ERG) have been implicated in repolarization of the cardiac action potential and also as components of the resting membrane conductance in various cells. The aim of the present study was to determine whether ERG channels were expressed in smooth muscle cells isolated from portal vein. RT-PCR demonstrated the expression of murine ERG (mERG), and real-time quantitative PCR showed that the mERG1b isoform predominated over the mERG1a, mERG2, and mERG3 in portal vein. Single myocytes from portal vein displayed membrane staining with an ERG1-specific antibody. Whole cell voltage-clamp experiments were performed to determine whether portal vein myocytes expressed functional ERG channels. Large inward currents with distinctive kinetics were elicited that were inhibited rapidly by E-4031 (mean amplitude of the E-4031-sensitive current at -120 mV was -205 +/- 24 pA; n = 14). Deactivation of the E-4031-sensitive current was voltage dependent (mean time constants at -80 and -120 mV were 103 +/- 9 and 33 +/- 2 ms, respectively; n = 13). Because of the rapid kinetics of mERG currents at more negative potentials, there was a substantial noninactivating "window" current that reached a maximum of -66 +/- 10 pA at -70 mV. Complete portal veins exhibited spontaneous contractile activity in isometric tension experiments, and this activity was modified significantly by E-4031. These data show that ERG channels are expressed in murine portal vein myocytes that may contribute to the resting membrane conductance.

Action Potentials↗