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Results for “mediation analysis”

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PubMed2004

Temperature-mediated heteroduplex analysis for detection of pncA mutations associated with pyrazinamide resistance and differentiation between Mycobacterium tuberculosis and Mycobacterium bovis by denaturing high- performance liquid chromatography.

The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in Mycobacterium tuberculosis isolates and simultaneously differentiate between M. tuberculosis and Mycobacterium bovis. Features that contributed to an optimal assay included the use of two different reference probes for the pncA gene targets from wild-type M. tuberculosis and wild-type M. bovis, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type M. tuberculosis strains (13 were PZA-resistant strains) and 21 M. bovis strains (8 were BCG strains). In all isolates tested, wild-type M. tuberculosis generated a single-peak pattern when mixed with the M. tuberculosis probe and a double-peak pattern with the M. bovis probe. In contrast, all M. bovis isolates generated a double-peak pattern when mixed with the M. tuberculosis probe and a single-peak pattern with the M. bovis probe. PZA-resistant mutant M. tuberculosis isolates generated characteristic patterns that were easily distinguishable from both wild-type M. tuberculosis and M. bovis isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between M. tuberculosis and M. bovis. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.

Animals↗

PubMed2005

High-resolution characterization of the canine DLA-DRB1 locus using reference strand-mediated conformational analysis.

Several methods exist for genotyping class II DLA gene polymorphisms in the dog. The most accurate method is sequence-based typing, which involves direct sequencing of polymerase chain reaction products. However, this method is expensive and unsuitable for large-scale studies. Recently, reference strand-mediated conformation analysis (RSCA) has been shown to be effective for characterizing major histocompatibility complex genes in humans, sheep, horse, and cats. RSCA is a cheap and rapid method, ideal for large epidemiological studies. We have developed RSCA for typing DLA-DRB1 in the dog. Control panels including dogs typed by sequence-based typing and cloned major histocompatibility complex class II alleles in plasmids were used to establish migration patterns for each allele using 20 different fluorescent labeled references, of which 5 were selected to allow for clear identification and discrimination of all known DLA-DRB1 alleles. We have compared 168 dogs typed by RSCA for DLA-DRB1 and characterized by sequence-based typing, with less than 1% discrepancy. These differences were due to missing alleles because of a weak polymerase chain reaction. To date, we have RSCA-typed 1,394 dogs. RSCA is likely to become the method of choice for characterizing DLA genes in the dog and will prove a useful tool for dissecting the immune response of dogs in clinical studies.

Alleles↗

PubMed2002

Frequency of HLA-DPB1 disparities detected by reference strand-mediated conformation analysis in HLA-A, -B, and -DRB1 matched siblings.

The role of HLA-DPB1 as transplantation antigen is controversial. The frequency and relevance of HLA-DPB1 mismatch in hematopoietic stem cell transplantation are unknown. To ascertain the rate of HLA-DBP1 mismatch in siblings that had been matched for HLA-A, -B, and -DRB1, reference strand mediated conformation analysis (RSCA) a high resolution HLA typing method was used. Locus-specific primers were used to amplify the HLA-DPB1 locus. The PCR product was then hybridized with two fluorescein-labeled references and the duplexes were analyzed after electrophoresis in a short polyacrylamide gel. Among the 113 pairs of individuals tested, six HLA-DPB1 mismatches were identified, which corresponds to a frequency of 5.31 % (95% confidence interval 3.20%-7.42 %).

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

HLA-B44 subtyping in the Catalan population using reference strand mediated conformation analysis. Implications for the selection of unrelated bone marrow donors.

Cytotoxic T lymphocytes (CTLs) reactive against the disparity between HLA-B*4402 and HLA-B*4403 have been reported after unrelated donor bone marrow transplantation. These CTLs have been associated with acute graft-versus-host disease and graft rejection. This study describes the HLA-B44-subtyping in the Catalan population using reference-strand mediated conformation analysis. It has been performed on 297 unrelated HLA-B44+ cord blood units from the Barcelona Cord Blood Bank (Barcelona, Spain). We have found a predominance of HLA-B*4403 (66.04%) over HLA-B*4402 (33.02%), whereas the predominant HLA-B44 allele in Northern Europe and the United States is HLA-B*4402. This inverted proportion between HLA-B44 subtypes in Mediterranean populations compared with other Caucasian populations suggests that HLA-B44 subtyping should be performed when an HLA-B44+ unrelated donor marrow is identified.

Alleles↗

PubMed2000

Genomic typing of minor histocompatibility antigen HA-1 by reference strand mediated conformation analysis (RSCA).

Disparities in minor histocompatibility antigen (mHAg) HA-1 are involved in the development of acute graft-versus-host disease (GvHD) in adult recipient after HLA-identical sibling donor hematopoietic stem cell transplantation. The mHAg HA-1 is an HLA-A*0201-restricted nonapeptide, which derives from the cleavage of a protein encoded at chromosome 19. The sequence analysis of HA-1 cDNA identified two alleles, termed HA-1H and HA-1R, which differ in only two nucleotides at 3' end of exon A, at positions 500 and 504. DNA-based methods for HA-1 typing were developed in 1998, using polymerase chain reaction with sequence-specific primers (PCR-SSP) and restriction fragment length polymorphism (PCR-RFLP). Here, we report the usefulness of reference strand mediated conformation analysis (RSCA), which was developed for mutation detection and typing of polymorphic loci, to discriminate between the two HA-1 alleles. We performed genomic typing of HA-1 locus in 203 HLA-A*0201-positive samples using RSCA and we confirmed these results by PCR-SSP. The results demonstrate the high reproducibility of this method and their strong correlation with the results obtained by PCR-SSP (99%). Only two samples showed disparity between the RSCA typing and the PCR-SSP. Direct sequencing of these samples confirmed that the correct allele assignment was that obtained by the RSCA typing. Furthermore, HA-1- RSCA-based typing provides additional information about the intronic structure of both alleles. With this approach, we describe the almost constant presence (99.2%) of a 5-bp deletion at intronic position 214-218 associated to the HA-1H allele, previously unidentified. We conclude that HA-1 genomic typing by RSCA is easy to perform and that could be used as a routine typing method.

Alleles↗

PubMed2026

Integrative cross-tissue transcriptome-wide association and metabolomic analysis reveals novel genetic risk loci for aortic aneurysm.

BACKGROUND: Aortic aneurysm (AA) is a life-threatening cardiovascular condition with a strong genetic component, however, its molecular mechanisms remain poorly understood. Although genome-wide association studies (GWAS) have identified numerous risk loci, most prior studies have investigated genetic and metabolic factors separately, leaving the causal pathways from genetic variants to disease largely unexplored. METHODS: We established an integrative framework combining cross-tissue transcriptome-wide association studies (TWAS) with metabolomic mediation analysis. First, we integrated GWAS data from FinnGen R12 with multi-tissue expression quantitative trait loci (eQTL) data from Genotype-Tissue Expression Project (GTEx) V8, then performed cross-tissue TWAS using the Unified Test for MOlecular SignaTures (UTMOST) and single-tissue validation with the Functional Summary-based Imputation (FUSION) to prioritize susceptibility genes. Second, we applied Mendelian randomization (MR), colocalization, and Fine-mapping Of CaUsal gene Sets (FOCUS) to assess causality and identify high-confidence genes. Third, we performed metabolite mediation analysis to uncover metabolic pathways linking genetic variants to disease risk. Finally, we validated key findings in mouse models of thoracic aortic aneurysm (TAA) and abdominal aortic aneurysm (AAA) using Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (RT-qPCR) and Western blotting. RESULTS: We identified multiple novel susceptibility genes for AA and its subtypes. Key genes included ADH family members (ADH1A, ADH1B, ADH4, ADH6) and ZNF827, which showed cross-subtype associations with strong colocalization evidence in vascular tissues. Metabolite mediation analysis revealed significant pathways involving N-acetylphenylalanine and methionine sulfoxide. Functional enrichment revealed distinct biological mechanisms: AA and AAA were primarily associated with metabolic pathways, whereas TAA-related genes were enriched in developmental and contractile processes. PheWAS indicated no significant off-target associations. Critically, experimental validation in mouse models confirmed significant upregulation of ZNF827 in TAA and ADH6 in AAA at both mRNA and protein levels, corroborating the genetic predictions. CONCLUSION: This integrated cross-omics analysis identifies novel genetic loci and, crucially, uncovers specific nutrient-related metabolic pathways that mediate genetic risk. These findings provide a mechanistic basis for future nutritional and metabolic intervention studies in AA and its subtypes.

MAGMA↗

PubMed2004

The use of reference strand-mediated conformational analysis for the study of cheetah (Acinonyx jubatus) feline leucocyte antigen class II DRB polymorphisms.

There is now considerable evidence to suggest the cheetah (Acinonyx jubatus) has limited genetic diversity. However, the extent of this and its significance to the fitness of the cheetah population, both in the wild and captivity, is the subject of some debate. This reflects the difficulty associated with establishing a direct link between low variability at biologically significant loci and deleterious aspects of phenotype in this, and other, species. Attempts to study one such region, the feline leucocyte antigen (FLA), are hampered by a general reliance on cloning and sequencing which is expensive, labour-intensive, subject to PCR artefact and always likely to underestimate true variability. In this study we have applied reference strand-mediated conformational analysis (RSCA) to determine the FLA-DRB phenotypes of 25 cheetahs. This technique was rapid, repeatable and less prone to polymerase chain reaction (PCR)-induced sequence artefacts associated with cloning. Individual cheetahs were shown to have up to three FLA-DRB genes. A total of five alleles were identified (DRB*ha14-17 and DRB*gd01) distributed among four genotypes. Fifteen cheetahs were DRB*ha14/ha15/ha16/ha17, three were DRB*ha15/ha16/ha17, six were DRB*ha14/ha16/ha17 and one was DRB*ha14/ha15/ha16/ha17/gd01. Sequence analysis of DRB*gd01 suggested it was a recombinant of DRB*ha16 and DRB*ha17. Generation of new alleles is difficult to document, and the clear demonstration of such an event is unusual. This study confirms further the limited genetic variability of the cheetah at a biologically significant region. RSCA will facilitate large-scale studies that will be needed to correlate genetic diversity at such loci with population fitness in the cheetah and other species.

Acinonyx↗

PubMed2002

Temperature-mediated heteroduplex analysis performed by using denaturing high-performance liquid chromatography to identify sequence polymorphisms in Mycobacterium tuberculosis complex organisms.

PCR products containing sequence polymorphisms were prepared from six mycobacterial genes, denatured, mixed with reference PCR products, and reannealed; the mixtures were then examined with a denaturing high-performance liquid chromatography system (WAVE) equipped with a temperature-controlled alkalated polystyrene divinyl benzene column. Mismatching of bases in heteroduplexes of the PCR products causes elution patterns of the DNA from the column to be altered. The six mycobacterial genes studied were oxyR, in which a specific polymorphism (G(1031)A) is found only in certain species of the Mycobacterium tuberculosis complex, and five genes in which mutations associated with antituberculosis drug resistance have been found. The resistance genes (with affected drug and PCR product sizes given parenthetically) were rpoB (rifampin; 258 bp), katG (isoniazid; 205 bp), pncA (pyrazinamide; 579 bp); rpsL (streptomycin; 196 bp), and embB (ethambutol; 185 bp). Elution patterns of heteroduplexes of all 20 polymorphisms studied shifted detectably at column temperatures ranging from 65.3 to 68 degrees C and elution times of 3.5 to 6 min. These results show that temperature-mediated heteroduplex analysis is a potentially useful genotypic screen for mutations associated with antituberculosis drug resistance and for the G(1031)A polymorphism in oxyR. The method may allow users to detect novel as well as heterogeneous mutations without using expensive kits or detection labels.

Antitubercular Agents↗

PubMed2026

The association of cardiovascular health with new-onset pulmonary hypertension and the mediating role of proteomic signatures.

BACKGROUND: The cardiovascular health (CVH) metrics have been reported to play an important role in the development of noncommunicable chronic diseases, yet its link to pulmonary hypertension (PH) risk and the underlying biological mechanisms remain unclear. This study aimed to investigate the association of CVH with PH risk and elucidate the mediating role of plasma proteomic signatures. METHODS: A total of 279 220 participants without PH at enrollment of the UK Biobank were included. Cox regression was used to quantify the association between CVH and incident PH. Proteome-wide association analysis, mediation analysis, and functional enrichment analysis were conducted to identify protein mediators. Key hub proteins were further validated at the transcriptional level through quantitative polymerase chain reaction (qPCR) in an animal model of PH, as well as at the protein level, and by macrophage-specific knockdown of interleukin (IL)-6 and CCL4 to evaluate its impact on rat pulmonary artery smooth muscle cell (PASMC) migration and proliferation. RESULTS: Over a median 13.2-year follow-up, 1325 PH cases occurred. Compared to the lowest CVH, participants with moderate and high CVH had 59% [hazard ratio (HR): 0.41; 95% confidence interval (CI): 0.33-0.49] and 82% (HR: 0.18; 95% CI: 0.14-0.23) lower risk, respectively. Proteomic analyses revealed that this association was significantly mediated by a distinct plasma protein signature. Pathway enrichment analysis indicates that proteins are significantly enriched in inflammatory/immune pathways, and key hub proteins were identified as participating in the central mechanism pathway. In the lung tissue of PH rat models, the mRNA and protein expression levels of IL-6 and C-C motif chemokine ligand 4 (CCL4) were significantly elevated. Furthermore, functional assays demonstrated that knockdown of IL-6 or CCL4 in macrophages significantly attenuated the migration and proliferation of rat PASMCs in vitro. CONCLUSION: High CVH level, defined by Life's Essential 8 (LE8), is significantly linked to a reduced risk of developing PH. This protective effect is primarily mediated by a proteomic signature, revealing the role of signaling pathways such as cytokine-cytokine receptor interaction in the prevention of PH.

Hypertension, Pulmonary↗

PubMedDate not supplied

Bronchospasm induced in allergic patients by in vitro released mediators. II. Analysis of possible mechanisms.

In a previous report, it was shown that cultures of peripheral blood leukocytes, from allergic individuals, in the presence of antigen will produce bronchospasm inducing agents in the supernatants. In this study, the responsibility of several mediators was analyzed, i.e., histamine, IgE, prostaglandins E2 and F2 alpha and leukotrienes C4. The results failed to demonstrate a significant release of histamine. Measurable amount of IgE could be recovered from active supernatants. Following removal of IgE and IgE-antigen complexes, the immediate bronchospastic response disappeared without so many changes in the delayed response. PGE2 and PGF2 alpha could be demonstrated in the active supernatants, which could be blocked by indomethacin. No significant differences in the bronchial challenge could be demonstrated between the two samples. Samples contained significant amount of leucotrienes C4. Treatment of cultures with NDGA (cyclooxygenase inhibitor) blocked almost completely the bronchial response. Therefore, in this assay, IgE, IgE-antigen complexes and leukotrienes C4 seem to play a major role in the induction of bronchospasm in sensitized individuals. We are clearly aware that the mediators production in vitro may eventually reflect only part of the events taking place in vivo. Nevertheless, this model allows the analysis of several major mediators and eventually some of their interactions. The role of PAF is now under study.

Asthma↗

PubMed2026

Genetically Predicted Gene Expression and Circulating Metabolites Associated with Cervical High-Grade Squamous Intraepithelial Lesion: A Mendelian Randomization Study.

BACKGROUND: High-grade squamous intraepithelial lesion (HSIL) is a precancerous condition of the cervix. Identifying risk factors associated with HSIL and understanding their potential mechanisms may inform prevention strategies. This study aimed to investigate the associations of genetically predicted gene expression and circulating metabolites with HSIL risk using Mendelian randomization (MR). METHODS: We performed two-sample MR analysis to evaluate the associations of genetically predicted gene expression (eQTLGen consortium, N=31,684) and circulating metabolites (genome-wide association study [GWAS], N=8,299) with HSIL risk (FinnGen R12, N=293,218; 8,291 cases). Mediation analysis was conducted to explore whether metabolites might mediate the associations between genes and HSIL. Sensitivity analyses, including Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO), leave-one-out, and colocalization, were performed to assess the robustness of the findings. All GWAS data used in this study were derived from European-ancestry populations. RESULTS: Eleven genes showed significant associations with HSIL after false discovery rate (FDR) correction (q<0.05), including VWA7, PAX8, GUSBP1, IKZF3, PAX8-AS1, NFKBIL1 (interpret with caution due to an influential single nucleotide polymorphism [SNP]), ERBB2, COL11A2, SKIV2L, TCF19, and PGAP3. Eleven circulating metabolites were also significantly associated with HSIL. Mediation analysis suggested that two phospholipid metabolites (GCST90200685 and GCST90200692) might mediate a small proportion of the total protective association of COL11A2 with HSIL (1.46% and 1.45%, respectively), indicating that the protective association of COL11A2 is largely independent of these circulating metabolites. Colocalization analysis showed strong evidence of shared causal variants for eight genes (PP.H4>0.98), while COL11A2 showed weak evidence of colocalization (PP.H4=1.58×10-15). Functional enrichment analysis indicated that COL11A2-related genes were enriched in extracellular matrix (ECM)-receptor interaction and PI3K-Akt signaling pathways. CONCLUSION: This MR study identified 11 genes and 11 circulating metabolites associated with HSIL risk. Among these, COL11A2 showed a protective association that appeared to be largely independent of circulating phospholipid metabolites, suggesting potential local mechanisms. These findings provide genetic and metabolic clues for future studies on HSIL etiology.

COL11A2↗

PubMed1988

T-cell-mediated cytolysis: analysis of killer and target cell deformability and deformation during conjugate formation.

T-cell-mediated cytolysis is initiated by the formation of strong adhesions between killer and target cells. The present work was aimed at determining whether T lymphocytes might exert some mechanical stress on targets during the binding process. Target S194 myeloma cells were thus conjugated to cytotoxic T lymphocytes (CTLs) raised by mixed lymphocyte culture or a cloned lymphoid line that was no longer cytolytic (TG2OUA2). After incubation periods of various lengths, conjugates were processed for electron microscopy and micrographs were digitized for computerized analysis: the cell surface curvature (at the micrometre level) and rugosity (at the submicrometre level) were quantified in free and adhesion-involved regions. Also, the size of cell interaction areas and the distribution of intermembrane distances were measured. Finally, TG2OUA2 and target cells were aspirated into glass micropipettes with calibrated pressure in order to assay their resistance to deformation by mechanical forces. The following conclusions were suggested. (1) Formation of extensive contact zones (with a linear size of several micrometres) with tight intermembrane adhesion (more than 30% of the membrane contours in adhesive zones were separated by an apparent distance lower than 500 A) was essentially completed within less than one minute. (2) CTLs or TG2OUA2 cells were more villous than their targets, and they seemed to deform in adhesive zones in order to adapt to the target contour, rather than imposing some deformation on the target. This may be a general feature of actively adherent cells. (3) Some CTLs, but no TG2OUA2 cells, exhibited conspicuous protrusions extending towards the bound target. In this case, the target cell but not the CTL displayed markedly increased roughness in the adhesion area. (4) TG2OUA2 cells were more resistant to mechanical deformation than S194 target cells, in accordance with previous reports suggesting that the membrane of CTLs was more resistant than that of target tumour cells. Hence, CTLs might transiently impose mechanical stress on the target membrane during the course of lethal-hit delivery.

Animals↗

PubMed2024

Leisure television watching exerts a causal effect on gastroesophageal reflux disease: evidence from a two-step mendelian randomization study.

BACKGROUND: Previous studies have shown that physical activity (PA) and leisure sedentary behaviors (LSB, including leisure television watching) are linked to gastroesophageal reflux disease (GERD). However, the associations between PA/LSB and GERD remain controversial. In this study, we aimed to reveal whether these associations reflect causal relationships and reveal the potential mechanisms of these relationships using bidirectional and two-step Mendelian randomization (MR) analyses. METHODS: We obtained genome-wide association study (GWAS) summary statistics for PA/LSB, four common risk factors (including cigarettes smoked per day, alcoholic drinks per week, triglycerides, total cholesterol) and GERD from published GWASs. A bidirectional MR analysis was performed to identify causal relationships between PA/LSB and GERD. Then, a series of sensitivity analyses were performed to verify the robustness of the results. Finally, a mediation analysis via two-step MR was conducted to investigate any effects explained by common risk factors in these relationships. RESULTS: Genetically predicted per 1-SD increase in leisure time television watching significantly increased the risk of GERD in the bidirectional MR analysis (OR = 1.33; 95% CI: 1.14-1.56; P = 2.71 × 10- 4). Sensitivity analyses successfully verified the robustness of the causal relationship. Further mediation analysis showed that this effect was partly mediated by increasing cigarettes smoked per day, with mediated proportions of 18.37% (95% CI: 11.94-39.79%). CONCLUSION: Our findings revealed a causal relationship between leisure television watching and an increased risk of GERD, notably, the causal effect was partially mediated by cigarettes smoked per day. These findings may inform prevention and management strategies directed toward GERD.

Humans↗

PubMed1991

Production of singlet oxygen-derived hydroxyl radical adducts during merocyanine-540-mediated photosensitization: analysis by ESR-spin trapping and HPLC with electrochemical detection.

Activated oxygen species produced during merocyanine 540 (MC540)-mediated photosensitization have been examined by electron spin resonance (ESR) spin trapping and by trapping reactive intermediates with salicylic acid using HPLC with electrochemical detection (HPLC-EC) for product analysis. Visible light irradiation of MC540 associated with dilauroylphosphatidylcholine liposomes in the presence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO/.OH). Addition of ethanol or methanol produced additional hyperfine splittings due to the respective hydroxyalkyl radical adducts, indicating the presence of free.OH.DMPO/.OH formation was not significantly inhibited by Desferal, catalase, or superoxide dismutase (SOD). Production of DMPO/.OH was strongly inhibited by azide and enhanced in samples prepared with deuterated phosphate buffer (PB-D2O), suggesting that singlet molecular oxygen (1O2) was an important intermediate. When MC540-treated liposomes were irradiated in the presence of salicylic acid (SA), HPLC-EC analysis indicated almost exclusive formation of 2,5-dihydroxybenzoic acid (2,5-DHBA), with production of very little 2,3-DHBA, in contrast to .OH generated by uv photolysis of H2O2, which gave nearly equimolar amounts of the two products. 2,5-DHBA production was enhanced in PB-D2O and inhibited by azide, again consistent with 1O2 intermediacy. 2,5-DHBA formation was significantly reduced in samples saturated with N2 or argon, and such samples showed no D2O enhancement. Ethanol had no effect on 2,5-DHBA production, even when present in large excess. Catalase and SOD also had no effect, and only a small inhibition was observed with Desferal. DMPO inhibited 2,5-DHBA production in a concentration-dependent fashion and enhanced formation of 2,3-DHBA. We propose that 1O2 reacts with DMPO to give an intermediate which decays to form DMPO/.OH and free.OH, and that the reaction between 1O2 and SA preferentially forms the 2,5-DHBA isomer. This latter process may provide the basis for a sensitive analytical method to detect 1O2 intermediacy. Singlet oxygen appears to be the principle activated oxygen species produced during MC540-mediated photosensitization.

Chromatography, High Pressure Liquid↗

PubMed2000

Protein redox potential measurements based on kinetic analysis with mediated continuous-flow column electrolytic spectroelectrochemical technique. Application to TTQ-containing methylamine dehydrogenase.

Kinetic determination of protein redox potentials with a mediated continuous-flow column electrolytic spectroelectrochemical technique (CFCESET) is described. In this method, the redox state of the mediator is completely regulated by the continuous-flow column electrolysis, and the homogeneous redox reaction between the mediator and a protein sample in the column is monitored spectroscopically at the downstream of the column. The protein/mediator reaction is in the pseudo-first-order kinetics, and then the rate equation is analytically solved. The kinetic analysis provides the protein redox potential as well as the homogeneous rate constant. In the kinetic measurements, equilibration of the system within the column is not required, which allows the use of increased kinds of mediators. This method was successfully applied to quinoprotein methylamine dehydrogenase containing tryptophan tryptophylquinone (TTQ) as a prosthetic group. The kinetic aspect is also valuable for the thermodynamic analysis with the mediated CFCESET. The half-life time of the kinetics can be utilized to optimize the system for the attainment of the equilibrated state within the column and can provide the assurance that the system is in equilibrium.

Electrochemistry↗

PubMed2004

Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" PCR mediated SSCP analysis.

AIMS: To carry out a retrospective study, screening for mutations of the entire coding region of RB1 and adjacent intronic regions in patients with retinoblastoma. METHODS: Mutation screening in DNA extracts of formalin fixed, paraffin wax embedded tissues of 28 patients using combined "exon by exon" polymerase chain reaction mediated single strand conformational polymorphism analysis, followed by DNA sequencing. RESULTS: Eleven mutations were found in 10 patients. Ten mutations consisted of single base substitutions; 10 were localised in exonic regions (eight nonsense, one missense, and one frameshift) and another one in the intron-exon splicing region. Three novel mutations were identified: a 2 bp insertion in exon 2 (g.5506-5507insAG, R73fsX77), a G to A transition affecting the last invariant nucleotide of intron 13 (g.76429G>A), and a T to C transition in exon 20 (g.156795T>C, L688P). In addition, eight C to T transitions, resulting in stop codons, were found in five different CGA codons (g.64348C>T, g.76430C>T, g.78238C>T, g.78250C>T, and g.150037C>T). Although specific mutation hotspots have not been identified in the literature, eight of the 11 mutations occurred in CGA codons and seven fell within the E1A binding domains (codons 393-572 and 646-772), whereas five were of both types-in CGA codons within E1A binding domains. CONCLUSIONS: CGA codons and E1A binding domains are apparently more frequent mutational targets and should be initially screened in patients with retinoblastoma. Paraffin wax embedded samples proved to be valuable sources of DNA for retrospective studies, providing useful information for genetic counselling.

Brazil↗

PubMed1986

Different characteristics of allogeneic and trinitrophenyl-modified H-2-restricted cell-mediated lympholysis: analysis of differences in interleukin-2 dependency using an ontogenical approach.

Ontogenical development of in vitro allogeneic cell-mediated lympholysis (CML) and in vitro trinitrophenyl (TNP)-modified CML was studied mainly in correlation with helper cell activity, using C3H/HeN and C57BL/6 mice. Allogeneic and TNP-modified CML were not detected in the spleen of these mice at 1 week after birth. Allogeneic CML was detectable, in parallel with increases in age. This activity in 5-week-old mice was much the same as in the 8-week-old mice but the TNP-modified CML did not appear until 8 weeks after birth. Exogenous interleukin-2 (IL-2) induced sufficient activity of TNP-modified CML, even in spleen cells from 1-week-old mice, while the same treatment had a weak but significant effect on the induction of allogeneic CML in these same cells. Experiments on the mixed lymphocyte reaction (MLR) in the presence of exogenous IL-2 showed that lymphocyte proliferation in response to TNP-modified cells was higher than that in response to allogeneic cells. These results suggest different dependencies on IL-2 between allogeneic and TNP-modified killer precursor cells. Endogenous IL-2 production and proliferative response in MLR showed that helper cells contributing to the TNP-modified CML matured later, compared to allogeneic CML. Different sensitivities to IL-2 in two types of CML, in addition to different ontogenical developments, suggest that cytotoxic T lymphocytes to allogeneic cells differ quantitatively and qualitatively from TNP-modified H-2-restricted killer cells.

Aging↗

PubMed1996

Pilot test results comparing the All Stars program with seventh grade D.A.R.E.: program integrity and mediating variable analysis.

A pilot test of a new program, All Stars, was completed. Four variables known from prior research to mediate high-risk behaviors were measured: 1) personal commitment to avoid participating in high-risk behaviors, 2) ideals incongruent with high-risk behaviors, 3) bonding with prosocial institutions, and 4) conventional beliefs about social norms regarding high-risk behaviors. Compared to students who received the seventh grade D.A.R.E. program, students' who received the All Stars program had significantly better outcomes on each mediator. All Stars students also gave superior ratings to the program and their involvement in it.

Adolescent↗