Optimization of a method for simultaneous analysis of cell surface phenotype and cell cycle in human bone marrow and peripheral blood leukocytes by flow cytometry: the value of both internal and external standards

Three different methods for the simultaneous analysis of surface phenotype and DNA quantification were compared. One method, involving the fixation of cells in 70% ethanol, was convincingly superior, both with regard to the CV of the G0G1 peak and the intensity of the DNA labelling. Furthermore, the correlation between the surface antigen densities before and after fixation were high. Experiments evaluating the intraday and the interday variation of the DNA ratio (the mean channel of the G0G1 peak of the sample divided by the mean channel of the G0G1 peak of chicken erythrocytes), documented the former to be small, with S.D. values varying from 0.0 to 0.016, while the latter were considerably higher with S.D. values varying from 0.077 to 0.123. Since the intraday variation of the DNA ratio was consistently low and the interday variation strongly correlated to the position of the red fluorescence test beads, it was possible to minimize the interday variation of the DNA ratio, by calculating the DNA index as the ratio between the DNA ratio of the sample and that of an external control (buffy coat leukocytes). Analyzing normal bone marrow and calculating the DNA index (DI) on the basis of these ratios, the confidence limits of the DI were decreased by more than half the values obtained when DI calculation was based solely on an internal standard, thereby making subsequent ploidy determinations of patient samples more precise. We conclude that this setup of internal and external standards allows accurate determinations of DNA aneuploidy even in an assay where whole cells labelled for surface antigen and DNA content are analyzed.     

Independent modulation of horse airway smooth muscle by epithelium and prostanoids

The effects of epithelial removal and cyclooxygenase inhibition on contractions induced by exogenous acetylcholine (ACh) and electrical field stimulation (EFS) were evaluated in horse tracheal strips and bronchial rings. Epithelial removal potentiated the response to ACh but had no influence on the response to EFS. The effect of epithelial removal was not altered by pretreating the tissues with meclofenamate, a cyclooxygenase inhibitor. In trachealis strips, meclofenamate augmented contractions induced by EFS but not by ACh. In bronchial rings, meclofenamate augmented EFS-induced contraction to a greater extent than ACh-induced contraction. These effects of meclofenamate were epithelium-independent. We conclude that horse airway epithelium produces a relaxant factor that is not a prostanoid. Endogenous prostanoids originating from non-epithelial sites inhibit only cholinergic nerves in the trachea but both parasympathetic nerves and smooth muscle in the bronchi.     

DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-L-methionine

The methyltransferase of the EcoK type I restriction/modification system is trimeric, M2S1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor S-adenosyl-L-methionine. Our results indicate that the methyltransferase has two non-interacting S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3.60 (+/- 0.42) microM determined by equilibrium dialysis, or 2.21 (+/- 0.29) microM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that S-adenosyl-L-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to S-adenosyl-L-methionine binding. Gel retardation results show that the binding of S-adenosyl-L-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.     

Induction of hyperhydration in rats by IP loading with graded concentrations of NaCl solution

In April 1991, 91 hospitalized patients in Cairo were reported to the Egyptian Ministry of Health with botulism intoxication. To define the spectrum of illness and identify a food vehicle, 45 patients were interviewed and a case-control investigation was conducted among families of 5 hospitalized patients. Clinical specimens and specimens of implicated food were tested for toxin and cultured for Clostridium botulinum. Hospitalized patients had symptoms consistent with botulism; 18 (20%) of 91 reported patients died. Illness was associated with eating faseikh (uneviscerated, salted mullet fish; lower 95% confidence limit of odds ratio = 6.6, P < .001). All 5 case-families purchased faseikh from one shop. Very high levels of type E botulinal toxin were detected in faseikh reported to be purchased from the implicated shop; C. botulinum type E was isolated from cultures of clinical specimens and from the faseikh. This is the first documented outbreak of botulism in Egypt and the largest type E outbreak ever reported.     

Ornithine-delta-aminotransferase expression and ornithine metabolism in cultured epidermal keratinocytes: toward metabolic sink therapy for gyrate atrophy

There is now strong evidence that the chorioretinal degeneration associated with ornithine-delta-aminotransferase (OAT) deficiency is a consequence of hyperornithinemia. Therefore development of a metabolic system for clearing ornithine from the circulation is being pursued as a potential treatment. The skin is considered an attractive location for such a metabolic system because autologous cells can be safely and easily utilized. This study was undertaken to determine the ornithine metabolizing capacity of epidermal keratinocytes expressing normal and superphysiologic amounts of OAT. The data show that overexpression of OAT in keratinocytes cultured from a gyrate atrophy patient restores ornithine metabolism and results in a rate of ornithine disappearance from the medium that is significantly higher than the rate of disappearance from the medium bathing normal keratinocytes. In addition, OAT activity determined in soluble protein prepared from sonicates suggests that the capacity to maintain plasma ornithine within the normal range is contained within an accomplishable graft of keratinocytes overexpressing OAT. However, the actual rate of ornithine disappearance from the media was significantly less than predicted from enzyme activity assays. Following ornithine metabolite production by intact cells suggests that ornithine metabolism is limited primarily by clearance of downstream metabolites, as opposed to substrate delivery.     

Isolation of a pentraxin-like protein from rainbow trout serum

To investigate the outcome of Graves' thyrotoxicosis after antithyroid drug management, data from 81 patients, treated in Chang Gung Memorial Hospital at Taipei and Linkou from October 1981 to March 1990, were analyzed. The gender ratio of female to male was 59:22. The mean age of onset was 33.1 +/- 10.5(15-60) year-old. All the patients were treated with antithyroid drug (Thionamide group) for a duration of 11 to 63 months (mean +/- SD = 28.1 +/- 9.8 months). Forty of 81 patients (49.4%) were remained remission after up to 2 years of follow-up. Those patients relapse usually occurred within 2 years after discontinuation of treatment (34/41), and only one exceptional case relapsed after 3 years. Three conditions affected the relapse rate. Patients with larger goiter (grade II-III) and shorter duration of treatment (< 23 months) had a higher relapse rate than those-with smaller goiter (grade O-I) [29/46 vs. 12/35; chi 2 = 6.576, p = 0.010; p = 0.015 in stepwise logistic regression (LR)] and longer duration of treatment (> or = 23 months) (15/20 vs. 26/61; chi 2 = 6.316, p = 0.012; p = 0.020 in LR). Patients with higher pre-treated serum triiodothyronine (T3) level (T3 > or = 300 ng/dl) had a higher relapse rate than those with lower T3 level (T3 < 300 ng/dl) in univariate analysis (30/50 vs. 11/31, chi 2 = 4.601, p = 0.032), but no significant difference by LR (P = 0.094). Other clinical parameters including age, sex, past history, family history, thyroxine (T4) level, T3/T4 ratio, thyroid autoantibodies, staging of ophthalmopathy, responsiveness to thyrotropin-releasing hormone stimulation test at the end of treatment, and whether combined treatment with thyroxine had no significant difference between the relapse and remission groups. These data suggest: (a) patients with larger goiter (grade II-III had higher relapse rate; (b) most of the recurrent thyrotoxicosis patients relapsed within two years after drug withdrawal; (c) continuing treatment for more than twenty-three months produces better outcome; (d) patients with Graves' thyrotoxicosis should be followed up for at least three years after withdrawal of antithyroid drug.     

Psychological research on race differences.

Responds to the discussion by A. H. Yee et al (see record 1994-09250-001) of A. R. Jensen's (e.g., see record 1986-18910-001) research on racial differences in abilities and states that the references to Jensen's work are dated. Jensen notes more recent research (Jensen and P. A. Whang; see PA, Vol 81:1181 and 20927) and provides an explanation for his research focus on Black–White differences in cognitive abilities. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A new technique for on-line and off-line high speed computation

A novel technique for both online and offline computation is presented. With this technique, a reconstruction analysis in elementary particle physics, otherwise prohibitively long, has been accomplished. It will be used online in an upcoming Fermilab experiment to reconstruct more than 100000 events per second and to trigger on the basis of that information. The technique delivers 40 gigaoperations per second, has a bandwidth on the order of gigabytes per second, and has a modest cost. An overview of the program, details of the system, and performance measurements are presented