Flow cytometric determination of basophils in whole blood with n-propyl Astra Blue iodide

Within the past year, it has become apparent, in connection with its use on automatic flow cytometers, that the quality of commercially available Alcian Blue has significantly declined. A homologous series of alkylated (C1-C7) Astra Blue quaternary ammonium halides was prepared, characterized, and evaluated for the detection of basophils in whole blood. On the Technicon H6000 flow cytometer, the resolution of the basophil cluster from the main population of unstained white blood cells was found to depend on the chain length of the quaternizing alkyl group. Optimal basophil resolution was observed for the n-propyl derivative. Correlation of the new method vs Alcian Blue as the reference on the H6000 was expressed as follows: %Baso (Astra Blue) = 0.89% Baso (Alcian Blue) + 0.12% for 180 fresh whole blood samples. Within-run precision at a basophil differential count of 0.73% was characterized by SD = 0.11, identical to that obtained for Alcian Blue. Aqueous solutions of n-propyl Astra Blue iodide, in contrast to Alcian Blue, are thermally stable. Heating the reagent for 1 h at 100 degrees C did not alter solubility or cytochemical behavior. In contrast, parallel treatment of Alcian Blue yielded insoluble material by hydrolysis of the isothiouronium groups. The reagent for basophil detection comprises n-propyl Astra Blue iodide, lanthanum chloride, sodium chloride, Tween 20, and cetylpyridinium chloride. The Astra Blue derivatives were characterized by uv-vis, ir, percentage halide, paper chromatography, and 13C NMR.     

Enzyme-coding genes as molecular clocks: The molecular evolution of animal alpha-amylases

Summary We constructed a cDNA library for the beetle,Tribolium castaneum. This library was screened using a cloned amylase gene fromDrosophila melanogaster as a molecular probe. Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids. Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results forD. melanogaster alpha-amylases, along with published sequences for other alpha-amylases. The results show that animal alpha-amylases are highly conserved over their entire length. A borader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals. We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.     

A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators.

In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.     

Seasonal fluctuations of selected physiological characteristics of elite alpine skiers

The effects of heavy resistance training and jumping exercise were examined during the 1989–1990 season in 12 international level alpine skiers. The athletes were tested before, during, immediately after training and during the period off training (June, July, October 1989, April 1990). Their mechanical behaviour was investigated using firstly squat jumps performed without (SJ) or with low extra loads (20 kg, SJ_20kg) and high extra loads (equivalent to body mass on the shoulders, SJ_bm) and secondly 15–30 s continuous jumping. These tests allowed the assessment of explosive dynamic strength production (SJ and SJ_20kg), slow dynamic strength (SJ_bm) and maximal mechanical power (continuous jumping). The training adopted resulted in specific changes in neuromuscular performance; in fact all the variables studied showed a significant improvement (P<0.01) from the beginning compared to the end of training. The range of improvement was between 55.4% (SJ_bm) and 12.5% (average power during 15-s continuous jumping). The enhancement of SJ had become significant by July. Surprisingly, even when no strength or jumping training was performed during the competition period (November-April), no deterioration in the neuromuscular performance was observed, there being no significant difference between the test values obtained in October 1989 and April 1990. It was concluded that the demanding competition programme of alpine skiers may provide a training stimulus adequate to maintain the neuromuscular improvement induced by training throughout the competition season.     

Subarctic forest-tundra vegetation gradients: The sigmoid wave hypothesis

Abstract. Spatial changes in tree and upland tundra cover in response to a complex environmental gradient and to landscape factors were investigated in the high subarctic forest-tundra of NW Canada. Vegetation and terrain studies provided ground truth for a grid of 1314 air photos which covered 24 % of the Canadian high subarctic and some of the adjacent low subarctic and low arctic. Across the high subarctic, gradual spatial change in % cover of tree and upland tundra vegetation is typical at both high and low cover values, with more rapid change occurring at intermediate cover. Cover gradients of zonal tree and tundra vegetation in the forest-tundra region in general follow a sigmoid pattern. Tundra and tree patch sizes increase in area and variability with higher tundra and tree cover, respectively.     

A simplified microassay for serotonin: Modification of the enzymatic isotopic assay

A simplification of the enzymatic isotopic assay for serotonin is described, Serotonin is converted to [³H]melatonin by a two-step reaction: N-acetylation of serotonin using acetic anhydride, followed by O-methylation with the enzyme hydroxyindole O-methyltransferase (EC 2.1,1.4) and S-adenosyl- -[methyl-³H]methionine as methyl donor. The present assay avoids the use of unstable acetylating enzyme, rat liver N-acetyltransferase (EC2.3,1.5). Blank values are lowered considerably and the sensitivity is doubly increased. Two-tenths micromole of serotonin per 30 μl of sample in tissue homogenates can be measured.     

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal.     

Characterization of PfiT/PfiA toxin–antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction

Toxin–antitoxin (TA) systems are small genetic modules usually consisting of two elements—a toxin and an antitoxin. The abundance of TA systems among various bacterial strains may indicate an important evolutionary role. Pseudomonas aeruginosa, which can be found in a variety of niches in nature, is an opportunistic pathogen for various hosts. While P. aeruginosa strains are very versatile and diverse, only a few TA systems were characterized in this species. Here, we describe a newly characterized TA system in P. aeruginosa that is encoded within the filamentous Pf4 prophage. This system, named PfiT/PfiA, is a homologue of the ParE/YefM TA system. It is a type II TA system, in which the antitoxin is a protein that binds the toxic protein and eliminates the toxic effect. PfiT/PfiA carries several typical type II characteristics. Specifically, it constitutes two small genes expressed in a single operon, PfiT inhibits growth and PfiA eliminates this effect, PfiA binds PfiT, and PfiT expression results in elongated cells. Finally, we assigned a novel function to this TA system, where an imbalance between PfiT and PfiA, favouring the toxin, resulted in cell elongation and an increase in virion production.