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At least 955 records · Page 53Linked to original sources
PubMed2005

Combining restriction digestion and touchdown PCR permits detection of trace isoforms of histamine H3 receptor.

The conserved sequences of the mouse histamine H3 receptor at the potential alternative splice junctions suggest that the splice isoforms found in guinea pig, rat, human, and hamster may also be present in the mouse. However, the trace amount isoforms are hard to be detected by the regular PCR approach. In this paper, we report a method in which the unspliced long isoform is cut by restriction endonuclease so that the short isoforms can be amplified to detectable levels to confirm the existence of the splice isoforms of H3 receptor mRNA in the mouse. This method is applicable to the detection of trace amounts of splice isoforms that coexist with the long, more abundant isoforms.

Animals↗
PubMed2002

RT-PCR heteroduplex analysis permits differentiation of transgene and host gene expression in a transgenic animal model.

In transgenic animal models, the conservation of DNA sequences between the transgene and the host wild-type gene can complicate the evaluation of the expression of each gene. The potential for gene silencing may complicate matters further. Here we report the use of RT-PCR heteroduplex analysis to differentiate the expression of a transgene and its homologous wild-type, even when these genes are very similar in their respective DNA sequences. We designed RT-PCR primers to amplify identically sized 243-bp fragments within the DNA binding domain of the p53 gene from both human and mouse mRNA samples. Ten samples from human p53 (273H) transgenic mice and 10 samples from wild-type controls were tested. Heteroduplex bands were formed in all transgenic samples but were absent from all wild-type samples. In addition, RT-PCR heteroduplex analysis was able in one sample to differentiate a silenced transgene from its wild-type allele, without the assistance of sequencing or labeling. In summary, the RT-PCR heteroduplex analysis is easy to use and has the ability to screen a large number of samples in a short time. The RT-PCR heteroduplex analysis is especially useful for the detection of expression when a transgene and the host homologous endogenous allele are too conserved in sequence to design species-specific RT-PCR primers.

Animals↗
PubMed1996

Assessment of a technology that permits individualized scan delays on helical hepatic CT: a technique to improve efficiency in use of contrast material.

OBJECTIVE: We performed this study to assess the usefulness of a computer automated scan technology (CAST) for individualizing scan delay during helical CT to improve the efficiency of hepatic enhancement. SUBJECTS AND METHODS: We prospectively evaluated 183 patients who were randomized into five groups. Control patients received 100 or 150 ml of contrast material (320 mg I/ml) with a 60-sec delay between contrast injection at 3 ml/sec and scanning. CAST patients received 100, 125, or 150 ml. In our latter groups we used an hepatic enhancement threshold of 50 H over baseline to determine the optimum delay between contrast injection and scanning. For the intergroup comparisons, we measured the liver on baseline and enhanced helical CT scans at the upper, mid, and lower levels of the liver. RESULTS: The mean enhancement in patients who received 150 ml of contrast material was 70.7 +/- 19.4 H for the control group and 81.0 +/- 17.5 H for the CAST group (p < .05). Hepatic enhancement above 50 H was achieved in 84% of the control subjects compared with 100% of CAST subjects; more than 60 H hepatic enhancement was achieved in 73% of control subjects and in 89% of CAST subjects. The use of CAST software with 125-ml contrast doses provided enhancement equivalent to that of control subjects who received 150 ml of contrast material (mean enhancement in CAST subjects, 70.3 +/- 15.4 H). Enhancement above 50 H was reached in 98% of CAST and 84% of control patients. With 100 ml of contrast material, 24% of patients failed to initiate CAST, resulting in enhancement similar to control patients (CAST, 54.2 +/- 11.4 H; controls, 56.9 +/- 15.2 H). CONCLUSION: Using a contrast dose of 150 ml, CAST provided significantly increased hepatic enhancement than that achieved in control subjects with less variability. For equivalent hepatic enhancement, contrast doses could be decreased by 25 ml using CAST technology because it provides individualized scan delays.

Adult↗
PubMed2003

Transcription and translation inhibitors permit metamorphosis up to radiole formation in the serpulid polychaete Hydroides elegans haswell.

Settlement and metamorphosis in most well-studied marine invertebrates are rapid processes, triggered by external cues. How this initial environmentally mediated response is transduced into morphogenetic events that culminate in the formation of a functional juvenile is still not well understood for any marine invertebrate. The response of larvae of the serpulid polychaete Hydroides elegans to inhibitors of mRNA and protein synthesis was examined to determine if metamorphosis requires these molecular processes. Competent larvae of H. elegans were induced to metamorphose by exposing them to a bacterial film or a 3-h pulse of 10 mM CsCl in the presence of the gene-transcription inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) or the translation inhibitor emetine. When induced to metamorphose in the presence of either inhibitor, larvae of H. elegans progressed through metamorphosis to the point at which branchial radioles start to develop. DRB and emetine inhibited the incorporation of radiolabeled uridine into RNA and radiolabeled methionine into peptides, respectively, indicating that they were effective in blocking the appropriate syntheses. Taken together, these results indicate that the induction of metamorphosis in H. elegans does not require de novo transcription or translation, and that the form of the juvenile worm is achieved in two phases. During the first phase, larvae respond to the inducer by attaching to the substratum, secreting a primary tube, resorbing the prototroch cilia, undergoing caudal elongation, and differentiating the collar; once the collar is formed, they begin secreting the secondary, calcified tube. During the second phase, the small worm develops branchial radioles and begins to grow, requiring new mRNA and protein syntheses.

Animals↗
PubMed1995

Individual multilocus genotypes using microsatellite polymorphisms to permit the analysis of the genetic variability within and between Italian beef cattle breeds.

We investigated the genetic variability within and between cattle breeds. The polymorphisms of 17 microsatellites were studied in 220 unrelated animals belonging to four Italian beef cattle breeds (Chianina, Marchigiana, Romagnola, and Piemontese). Variations of allelic frequencies were examined to characterize the breeds and their relationships. Wahlund coefficients, Polymorphism Information Content values, and Haldane exact test for Hardy-Weinberg proportions were calculated. The results show that the Hardy-Weinberg equilibrium is not always maintained. Moreover, in addition to the classical genetic distances, a new method, based on the consideration of a multilocus genotype of each animal, was set up to measure the genetic similarity between animals or within groups of animals. All the results showed that, whereas Chianina occupies an intermediate position and Piemontese is the most distinct of all four breeds, Marchigiana and Romagnola display the strongest similarity. The new method also provides evidence that average similarities are always higher within breeds than between breeds. By comparing pairwise the multilocus genotypes, it was also possible to discriminate the individuals with higher or lower genetic similarities so that each breed could be subdivided into two groups of animals in relation to their similarity to the average breed multilocus genotype. High similarities between breeds were detected, somewhat surprisingly, when the most homogeneous groups of each breed were compared. The microsatellite multilocus genotype is particularly efficient in evaluating the between- and within-breeds genetic similarities and for subgrouping genetically more homogeneous animals.

Alleles↗
PubMed1997

Stress down-regulates experimental allergic encephalomyelitis (EAE) but permits activation and localization of autoreactive V beta 8.2+ T cells.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease inducible by encephalitogenic helper T cells expressing V beta 8.2. In this study, we examined the relationship between the stressor-induced alternation of clinical EAE and the induction of autoreactive T cells using Lewis rats. Animals were immersed for 5 min in a water bath maintained at 44 degrees C continuously for 10 or 13 days, before or after the immunization of the encephalitogenic peptide, respectively. Stress administrations after the immunization clearly diminished the severity of clinical EAE, and delayed the onset of disease. On the other hand, stress administrations prior to the immunization resulted in the marginal suppression of clinical EAE. Splenocytes of the stressed rats showed, however, comparative proliferative responses to the encephalitogenic peptide or mitogens with that of the control rats. Moreover, higher level of V beta 8.2 mRNA expression was detected in the spinal cords of the stressed rats than in control rats. Sequence analysis of CDR3 region of TCR cDNA showed that V beta 8.2+ T cells in the spinal cords of the stressed rats possess common features with the biased encephalitogenic T cells. These results suggest that the stressor-induced suppression of clinical EAE is not simply because of the failure of induction of autoreactive T cells, nor localization of the autoreactive T cells in the central nervous system.

Amino Acid Sequence↗
PubMed1995

Zaditen SRO permits once-daily dosing with superior efficacy in the prophylaxis of asthma.

This international, multicenter clinical trial was designed to compare the efficacy and safety of two different formulations of ketotifen: Zaditen SRO and Zaditen Standard Form. In a randomized double-blind study over a 12-week treatment period, 3 parallel groups of asthmatic subjects received Zaditen SRO (2 mg once daily), Zaditen SRO (4 mg once daily), or Zaditen Standard Form (1 mg twice daily). Asthmatic subjects (362 evaluable cases, aged 6-29 years) kept daily records of clinical symptoms, use of concomitant medication, and peak flow recordings and were examined at 2-week intervals up to the end of the study. Zaditen SRO 4 mg administered once a day at night showed a statistically significant faster onset of action and was more clinically effective than Zaditen Standard Form. The Zaditen SRO 4-mg and 2-mg formulations were at least as well tolerated as the standard form, with somnolence occurring equally after both formulations. In conclusion, Zaditen SRO (4 mg once daily) was found to be equally safe and more effective in the prophylactic treatment of mild and moderate bronchial asthma than Zaditen Standard Form (1 mg twice daily).

Adolescent↗
PubMed1997

Rapid denaturation improves chromosome morphology and permits multiple hybridizations during fluorescence in situ hybridization.

Denaturation of chromosomal DNA for fluorescence in situ hybridization (FISH) is an essential step in a procedure associated with a number of variables. In our experience, shorter denaturation time in 70% formamide/2 x SSC at 72 C provides sufficient denaturation, where the hydrogen bonds are broken between the purines and pyrimidines of the double helix. This shortened exposure improves retention of morphology of human chromosomes from lymphocytes, aminocytes, fibroblasts and bone marrow, and allows the same metaphases to be denatured repeatedly and rehybridized with different probes. This approach is useful in investigations where sample volume is limited.

Chromosomes, Human↗