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PubMed2006

Development of the high throughput screening assay for identification of agonists of an orphan nuclear receptor.

Retina-specific nuclear receptor (RNR), also known as PNR and NR2E3, is an orphan nuclear receptor expressed exclusively in photoreceptor cells of the retina. Here we describe homogeneous cell-based resonance energy transfer assay for identification of RNR agonists using beta-lactamase as the reporter gene. Bacterial beta-lactamase reporter construct containing GAL4 response elements was randomly integrated into the genome with subsequent selection of responsive cell pools by fluorescence-activated cell sorting. Chimeric RNR (RNR hinge and ligand-binding domains fused to GAL4 DNA-binding domain) was stably transfected into mammalian Flp-In Chinese hamster ovary cells using Flp-mediated recombination into a single pre-integrated Flp recombination target site. Since no RNR ligand could be used as a control for monitoring the development of the RNR assay, we developed a parallel cell line with the functionally related well-characterized thyroid hormone nuclear receptor. This parallel thyroid hormone nuclear receptor system was used as a "guide" in optimizing the RNR assay for ultra-high-throughput screening in 3,456-well nanoplate format. The assay was successfully used to screen a large compound collection for RNR agonists. In this study we demonstrated the feasibility of developing and optimization of the high-throughput screening-compatible assay for the orphan nuclear receptor in the absence of its cognitive ligand.

Animals↗
PubMed1993

Rheumatic heart disease in the developing world: prevalence, prevention, and control.

Rheumatic heart disease (RHD) continues to be a common health problem in the developing world, causing morbidity and mortality among both children and adults. Although little longitudinal data are available, evidence suggests that there has been little if any decline in the occurrence of RHD over the past few decades. Recent reports from the developing world have documented rheumatic fever (RF) incidence rates as high as 206/100,000 and RHD prevalence rates as high as 18.6/1000. The high frequency of RHD in the developing world necessitates aggressive prevention and control measures. The major interventions for prevention and control include: (1) reduction of exposure to group A streptococci, (2) primary prophylaxis to prevent initial episodes of RF, and (3) secondary prophylaxis to prevent recurrent episodes of RF. Because recurrent episodes of RF cause increasingly severe cardiac complications, secondary prophylaxis is the most crucial feature of an effective RHD programme. For some impoverished countries, secondary prophylaxis may be the only intervention that can realistically be implemented. In addition to this intervention, however, financial and human resources must be committed, and all of these elements must be integrated into existing primary health care systems. Because RHD continues to be a common health problem in the developing world, greater emphasis needs to be placed on the simple and cost-effective prevention and control measures that are currently available to combat this disabling disease.

Cross-Cultural Comparison↗
PubMed2005

Combined prognostic utility of ST-segment recovery and myocardial blush after primary percutaneous coronary intervention in acute myocardial infarction.

AIMS: ST-segment recovery (SigmaSTR) and myocardial blush (MB) evaluate different elements of microcirculatory integrity after reperfusion therapy in acute myocardial infarction (AMI). We sought to determine whether the combination of SigmaSTR and MB after primary percutaneous coronary intervention (PCI) in AMI has greater prognostic utility than either measure alone. METHODS AND RESULTS: The 30 days and 1 year clinical outcomes of 456 patients were assessed as a function of SigmaSTR and MB after primary PCI from the CADILLAC trial. SigmaSTR and MB were concordant (> or =70% SigmaSTR and MB grade 2/3 or <70% SigmaSTR and MB grade 0/1) in 60.1% of patients and discordant in 39.9% of patients. The greatest survival was observed among patients with complete SigmaSTR (> or =70%) and MB grade 2/3 in whom the cumulative rates of death at 30 days and 1 year were 0.6 and 1.2%, respectively. Poorest survival was observed among patients with incomplete SigmaSTR (<70%) and reduced MB (grade 0/1), in whom 30 days and 1 year rates of death were 8.3 and 10.1%, respectively. Intermediate outcomes were present in patients with discordant MB and SigmaSTR. By multivariable analysis, however, SigmaSTR was an independent correlate of survival at 30 days and 1 year (P=0.05 and 0.01, respectively), whereas MB was no longer predictive (P=0.38 and 0.72, respectively). CONCLUSION: SigmaSTR and MB are not infrequently discordant after primary PCI. By univariate analysis, both measures of reperfusion success strongly correlate with survival and assessment of both yields incremental prognostic information beyond either measure alone. By multivariable analysis, however, SigmaSTR is the stronger prognostic variable.

Aged↗
PubMed1995

Intermolecular transposition of IS10 causes coupled homologous recombination at the transposition site.

Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid. This was detected by the activity of the tet gene expressed from the phage 434 PR promoter. Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site. Cointegrate formation was abolished in delta recA strains, although simple insertions of IS10 were observed. This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor. Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event. Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome. This was accompanied by the formation of an additional copy of IS10 in the chromosome. Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10.

Base Sequence↗
PubMed1994

Differences in the tissue-specific homing of alpha beta and gamma delta T cells to gut and peripheral lymph nodes.

Tissue-selective streaming of T cells is considered to be a critical element in the integration of normal immune responses in intact animals. The results presented in this paper show that while there were major subsets of gut-homing T cells present in intestinal lymph, there were considerable differences in the tissue tropism of T cell populations circulating in lymph draining gut and peripheral lymph nodes. Thus, while CD4+ cells returned preferentially to their tissue of origin, gamma delta T cells showed a strong migratory preference for peripheral lymph nodes regardless of their tissue of origin. In contrast, although a population of gut-homing CD8+ cells was present in ileal lymph, CD8+ T cells from peripheral lymph nodes homed equally well to gut and lymph nodes. There were also considerable differences in the expression of L-selectin on T cells circulating in the two compartments. L-selectin was down-regulated on alpha beta T cells present in ileal lymph but not on gamma delta T cells which expressed the highest levels of L-selectin of all T cell subsets. It is suggested that gut-homing alpha beta T cells which have down-regulated L-selectin are formed in the gut-associated lymphoid tissues in response to gut antigens while the migratory properties of gamma delta T cells are ontogenetically determined, independent of antigen.

Animals↗
PubMed2003

From signal to cell polarity: mitogen-activated protein kinases as sensors and effectors of cytoskeleton dynamicity.

Mitogen-activated protein kinases (MAPKs) are ubiquitous phosphorylation enzymes involved in signal transduction, gene expression and activation of diverse cytoskeletal proteins. MAPKs participate in the regulation of a broad range of crucial cellular processes including cell survival, division, polarization, stress responses, and metabolism. Phosphorylation of cytoskeletal proteins usually results in the rearrangement of cytoskeletal arrays leading to morphological changes and cell polarization. On the other hand, some cytoskeletal motor proteins, such as kinesins, could activate MAPK members and participate in signal delivery to the proper cellular destination (e.g. during cell division). Moreover, changes in the integrity of cytoskeletal elements have direct impacts on MAPK activity. Recent evidence suggests that there is bi-directional signalling between MAPK cascades and cytoskeleton. The focus here is on this cross-talk between MAPK signalling and the cytoskeleton in various eukaryotic systems including yeast, plants, and mammals and a role is proposed for MAPKs as sensors monitoring the cytoskeleton-dependent balance of forces within the cell.

Animals↗
PubMed1990

Does butylphenyl-deoxyguanosine triphosphate differentially inhibit DNA polymerase alpha and delta activities in permeabilized HeLa cells?

In eukaryotic cells, two enzymes, DNA polymerases alpha and delta, are thought to play major roles in DNA synthesis. I have used butylphenyl dGTP (BuPdGTP), a potent inhibitor of purified DNA polymerase alpha, to assess the relative activities of these enzymes in two permeabilized cell systems. In both instances BuPdGTP eliminated all of the activity which was sensitive to aphidicolin. However, no conditions were found where BuPdGTP preferentially inhibited the synthesis of Okazaki fragments--the presumed products of DNA polymerase alpha activity. This implies that DNA polymerase activities on the two sides of the replication fork are unable to operate independently, being just two elements of the integrated replication machinery that undertakes DNA synthesis in permeabilized cells.

Cell Membrane Permeability↗
PubMed2001

Gene expression in isolated plant mitochondria: high fidelity of transcription, splicing and editing of a transgene product in electroporated organelles.

Mitochondrial gene expression was studied using an electrotransformation protocol to introduce foreign DNA into purified wheat mitochondria. Optimal conditions for DNA uptake and transient gene expression were determined. We show here that a DNA plasmid containing either a cognate or a non-cognate gene under the control of a plant mitochondrial promoter is incorporated into the organelle and faithfully recognized by the transcription machinery. Transcripts generated by a plasmid bearing the intron-containing cox II gene were correctly spliced. Moreover, the transcripts were edited at the expected target C residues. The expression and maturation process of the transgene is dependent on the integrity of functional elements such as the promotor or the presence of structural domains necessary for splicing. The mitochondrial transformation described in this report is an important tool to study the multiple steps involved in plant mitochondrial gene expression at conditions closer to those found in vivo.

Base Sequence↗
PubMed2006

Tumor inhibition by genomically integrated inducible RNAi-cassettes.

RNA interference (RNAi) has emerged as a powerful tool to induce loss-of-function phenotypes by post-transcriptional silencing of gene expression. In this study we wondered whether inducible RNAi-cassettes integrated into cellular DNA possess the power to trigger neoplastic growth. For this purpose inducible RNAi vectors containing tetracycline (Tet)-responsive derivatives of the H1 promoter for the conditional expression of short hairpin RNA (shRNA) were used to target human polo-like kinase 1 (Plk1), which is overexpressed in a broad spectrum of human tumors. In the absence of doxycycline (Dox) HeLa clones expressing TetR, that carry the RNAi-cassette stably integrated, exhibited no significant alteration in Plk1 expression levels. In contrast, exposure to Dox led to marked downregulation of Plk1 mRNA to 3% and Plk1 protein to 14% in cell culture compared to mismatch shRNA/Plk1-expressing cells. As a result of Plk1 depletion cell proliferation decreased to 17%. Furthermore, for harnessing RNAi for silencing disease-related genes in vivo we transplanted inducible RNAi-HeLa cells onto nude mice. After administration of Dox knockdown of Plk1 expression was observed correlating to a significant inhibition of tumor growth. Taken together, our data revealed that genomically integrated RNAi-elements are suitable to hamper tumor growth by conditional expression of shRNA.

Animals↗
PubMed2002

Elimination of blinding trachoma.

PURPOSE OF REVIEW: A recently initiated major effort to eliminate blinding trachoma as a public health problem is discussed. RECENT FINDINGS: Mass (community-wide) treatment with a short course of oral azithromycin has been shown to be at least as effective as long courses (42 doses over a 6-week period) of topical tetracycline in reducing clinically active trachoma and the prevalence of chlamydial infection. The beneficial effects last for more than a year, in contrast to the short-lived results typically seen after previous control programs that used topical therapy. Azithromycin is a key element in the integrated approach towards eliminating blinding trachoma summarized as the 'SAFE' strategy (surgery for deformed eyelids; antibiotic treatment of whole communities with azithromycin, to control infection; face washing and improved hygiene of young children; and environmental improvements, particularly the provision of water and latrines). The first regional application of this strategy has shown dramatic results, producing a reduction in trachoma activity of more than 70% in Morocco. SUMMARY: The goal of eliminating blinding trachoma may be reachable. Further research is needed to determine how best to use azithromycin and which environmental improvements will be most effective. The important questions are as follows: how often antibiotic treatment programs will be required in the endemic areas; how best to evaluate the effects of treatment; and when (and perhaps whom) do we retreat.

Anti-Bacterial Agents↗
PubMed1999

Environmental health impact assessment: evaluation of a ten-step model.

"Environmental impact assessment" denotes the attempt to predict and assess the impact of development projects on the environment. A component dealing specifically with human health is often called an "environmental health impact assessment." It is widely held that such impact assessment offers unique opportunities for the protection and promotion of human health. The following components were identified as key elements of an integrated environmental health impact assessment model: project analysis, analysis of status quo (including regional analysis, population analysis, and background situation), prediction of impact (including prognosis of future pollution and prognosis of health impact), assessment of impact, recommendations, communication of results, and evaluation of the overall procedure. The concept was applied to a project of extending a waste disposal facility and to a city bypass highway project. Currently, the coverage of human health aspects in environmental impact assessment still tends to be incomplete, and public health departments often do not participate. Environmental health impact assessment as a tool for health protection and promotion is underutilized. It would be useful to achieve consensus on a comprehensive generic concept. An international initiative to improve the situation seems worth some consideration.

Environmental Exposure↗
PubMed1996

Detection of Mycobacterium tuberculosis in formaldehyde solution-fixed, paraffin-embedded tissue by polymerase chain reaction in Pott's disease.

STUDY DESIGN: Twenty-five formaldehyde solution-fixed, paraffin-embedded tissue blocks from vertebral biopsy specimen materials with presumptive diagnosis of tuberculous spondylitis and nonspecific vertebral osteomyelitis were studied. OBJECTIVES: To evaluate the sensitivity and specificity of polymerase chain reaction in detecting Mycobacterium tuberculosis in formaldehyde solution-fixed, paraffin-embedded tissue samples from histologically proved tuberculous spondylitis. SUMMARY OF BACKGROUND DATA: Diagnosis of a mycobacterial infection is a long and tedious process; because of the slow growth rate of mycobacteria on solid media, identification and antibiotic sensitivity testing can take up to 10 weeks, but the sensitivity of culture can be as low as 50%. Direct microscopy is insensitive because clinical samples may contain only few organisms. Recently, polymerase chain reaction has been applied in the rapid amplification and identification of many organisms, including mycobacteria. METHODS: The DNAs were extracted from 25 paraffin-embedded tissue blocks. An insertion element IS 6110 (Integrated DNA Tec. Inc., Corrallville, IA), a DNA sequence unique to Mycobacterium complex (M. tuberculosis and the subspecies Mycobacterium bovis), was amplified by polymerase chain reaction. Polymerase chain reaction results were compared with those of Mycobacterium culture, acid-fast bacilli staining, and histologic findings. RESULTS: Polymerase chain reaction was positive in 18 cases of 19 tuberculous spondylitis. Three of the polymerase chain reaction test results were positive with concomitant negative culture and positive acid-fast bacilli staining. There were six chronic nonspecific infections, and polymerase chain reaction results were negative in five cases; in the single positive case, DNA amplification results remained positive even after three repeated tests. CONCLUSION: Polymerase chain reaction has a sensitivity of 94.7%, specificity of 83.3%, positive predictive value of 94.7%, and a negative predictive value of 83.3%. Accuracy was calculated as 92%.

Fixatives↗
PubMed1980

Transfer of gentamicin resistance between cultures of Staphylococcus aureus in nutrient broth, serum and urine.

Only one of 23 gentamicin-resistant cultures of Staphylococcus aureus transferred its resistance in mixed culture in broth to the non-lysogenic S. aureus strain 1030; the transfer-frequency was 10(-3)-10(-4). Transfer between non-lysogenic clones of strain 1030 occurred at a similar frequency in urine, and at a frequency of approximately 10(-1) in serum. The resistance determinant for transfer between non-lysogenic clones was usually linked to phage genome, as shown by the possession by resistant recipients of immunity to typing phage 75, plaque-forming particles in their culture filtrates and inducibility by mitomycin C. Stability of the resistance on storage and transduction kinetics suggested that these genes were chromosomal. Two resistant derivatives were isolated that had lost some phage functions and were unable to transfer their resistance further. The epidemiology of gentamicin resistance may in part be explicable by the transient formation of an auto-transmissible element with subsequent integration of the resistance genes into a variety of replicons (i.e., transposition).

Blood↗
PubMed1989

The Hox-1.3 homeo box protein is a sequence-specific DNA-binding phosphoprotein.

We report that the murine Hox-1.3 homeo domain protein is a nuclear phosphoprotein capable of binding to specific DNA sequences. DNase I protection of the Hox-1.3 gene promoter region with the Hox-1.3 protein identifies a binding site 144 bp upstream from the start of transcription. Both phosphorylated and nonphosphorylated forms bind DNA directly in a sequence-specific manner. Electrophoretic mobility shift assays were performed with a set of synthetic oligonucleotides representing either the DNase I-protected region of the Hox-1.3 gene or partially homologous sequences present in promoter regions of other characterized viral, yeast, and mammalian genes. From the results, we deduce a consensus binding motif of CPyPyNATTAT/GPy. Base substitutions in the core ATTA sequence severely reduce or abolish binding. In the SV40 enhancer, the Hox-1.3 binding motif overlaps both the octamer (Octa2) and the transactivator protein-1 (AP-1) binding sites. The Hox-1.3 binding motif also overlaps the nuclear factor III (NF-III) octamer motif in the adenovirus-2 origin of DNA replication. Overlap among DNA-binding sites suggests that regulation imparted by certain cis-elements may be integrated by these different factors.

Adenoviridae↗
PubMed2005

Retrotransposon accumulation and satellite amplification mediated by segmental duplication facilitate centromere expansion in rice.

The abundance of repetitive DNA varies greatly across centromeres within an individual or between different organisms. To shed light on the molecular mechanisms of centromere repeat proliferation, we performed structural analysis of LTR-retrotransposons, mostly centromere retrotransposons of rice (CRRs), and phylogenetic analysis of CentO satellite repeats harbored in the core region of the rice chromosome 4 centromere (CEN4). The data obtained demonstrate that the CRRs in the centromeric region we investigated have been enriched more significantly by recent rounds of segmental duplication than by original integration of active elements, suggesting that segmental duplication is an important process for CRR accumulation in the centromeric region. Our results also indicate that segmental duplication of large arrays of satellite repeats is primarily responsible for the amplification of satellite repeats, contributing to rapid reshuffling of CentO satellites. Intercentromere satellite homogenization was revealed by genome-wide comparison of CentO satellite monomers. However, a 10-bp duplication present in nearly half of the CEN4 monomers was found to be completely absent in rice centromere 8 (CEN8), suggesting that CEN4 and CEN8 may represent two different stages in the evolution of rice centromeres. These observations, obtained from the only complex eukaryotic centromeres to have been completely sequenced thus far, depict the evolutionary dynamics of rice centromeres with respect to the nature, timing, and process of centromeric repeat amplification.

Centromere↗
PubMed1975

Mechanism of induction of RNA tumor viruses by halogenated pyrimidines.

Frome these studies on JLS V-9 cells, a number of conclusions can be drawn about the mechanism of MuLV induction by halogenated pyrimidines. The compounds can induce virus from otherwise healthy cells as long as deoxycytidine is present along with the inducing agent. The compounds must be present during the S phase of the cell cycle and must be incorporated into DNA in order to induce virus (Teich et al. 1973). Only one strand of DNA need be substituted by BrdU or IdU in order to induce virus, because a one-hour period of incorporation leads to induction. From these results it is possible to construct a model for how halogenated pyrimidines are able to induce viruses from otherwise uninfected cells. Because the critical period for the incorporation of the compound is a restricted segment of the S phase of the cell, there would appear to be a critical segment of the genetic information of the cell which, when substituted with BrdU or IdU, leads to a transcriptional derepression. Presumably the critical segment of DNA is either a controlling element of the integrated provirus or it is a separate gene which controls the expression of the integrated provirus. Whichever is true, these results strongly imply that the search for specific repressors of the segments of mammalian DNA is likely to be successful and that RNA tumor viruses may offer a system in which such repression systems can be identified and investigated.

Bromodeoxyuridine↗
PubMed1990

The integration-excision system of the conjugative transposon Tn 1545 is structurally and functionally related to those of lambdoid phages.

Excision of Tn1545 and related conjugative transposons of Gram-positive bacteria occurs by reciprocal site-specific recombination between non-homologous regions of the transposon-target junctions. Excisive recombination requires two transposon-encoded proteins designated Xis-Tn and Int-Tn. We have shown that, following excision, Tn1545 is a circular structure with ends separated by either of the two hexanucleotides that were present at the transposon-target junctions. Using a trans-complementation assay, we have demonstrated that Int-Tn is able to catalyse in vivo integration of a circular intermediate of Tn1545 defective for integration and excision. comparison of integration sites suggests that limited sequence homology at the vicinity of the recombining sites is required for integration of the element. These data support the hypothesis that the integration/excision systems of conjugative transposons from Gram-positive cocci and of lambdoid phages from Gram-negative bacilli have evolved from a common ancestor.

Bacteriophage lambda↗
PubMed1990

Gas supply across membranes in bioreactors for hepatocyte culture.

The conditions required for hepatocyte cultures is a main topic in the development of bioreactors for hybrid liver support systems. The detoxification of ammonia and the synthesis of urea due to primary isolated hepatocytes was measured in order to compare two different models of gas supply in bioreactors: (a) indirect medium oxygenation and (b) direct membrane-contact oxygenation of the hepatocytes using polypropylene membranes. Increasing oxygen pressure promoted cell function. At day 6 of culture, urea synthesis was 0.8 +/- 0.3 mM in 21% of O2 cultures and 1.5 +/- 0.1 mM in oxygenated cultures. Alkalosis due to CO2 loss decreased ammonia metabolism. The direct membrane-contact oxygenation resulted in enhanced cell metabolism in comparison to medium oxygenation: urea synthesis at day six was 1.42 +/- 0.2 mM in 21% O2 cultures. Polypropylene oxygenation membranes proved to be sufficient for hepatocyte adhesion. Two functions can be integrated in one element in liver support systems using the investigated polypropylene membrane and the direct membrane-contact oxygenation: oxygenation with physiological oxygen pressure in bioreactors due to gas supply across the membrane and adhesion of hepatocytes in bioreactors on the membrane.

Animals↗