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.     

Phylogenetic and biochemical characterization of acidophilic bacteria

Abstract: In the course of our studies to determine appropriate conditions to transform Thiobacillus ferrooxidans , we have adopted a tetrathionate-containing medium. A number of different, apparently stable colony morphologies have arisen from cultures presumed to be pure while grown on iron-containing medium. In an effort to assess whether these different morphologies are indicative of previously undetected contamination, or simply manifestations of physiological variability within a strain, we have applied a number of biochemical and genetic techniques, including cellular fatty acid analysis and 16S rRNA sequence determination. These data should allow us to unequivocally characterize and compare the organisms present in our cultures.     

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.     

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.     

Modeling force application configurations and morphologies required for cancer cell invasion

Biomechanics and Modeling in Mechanobiology - We show that cell-applied, normal mechanical stresses are required for cells to penetrate into soft substrates, matching experimental observations in...     

Superiority of Trans-Oral over Trans-Nasal Sampling in Detecting Streptococcus pneumoniae Colonization in Adults

The human nasopharynx is the main reservoir for Streptococcus pneumoniae. We applied conventional and molecular methods to determine the prevalence of S. pneumoniae nasopharyngeal colonization in adults. Paired trans-orally and trans-nasally obtained nasopharyngeal samples from 268 parents of 24-month-old children were assessed for pneumococcal presence. Parents were classified as colonized when live pneumococci were recovered from either sample cultured on medium selective for S. pneumoniae. Of the 52 (19%) colonized parents 49 (18%) were culture-positive in trans-nasal and 10 (4%) in trans-oral samples. Bacterial growth was harvested from these cultures, DNA isolated and tested by quantitative-PCR (qPCR) targeting lytA and piaA genes specific for S. pneumoniae. A sample was considered positive if signals for both genes were detected. Altogether 105 (39%) individuals were classified as positive for pneumococcus by qPCR including 50 (19%) in trans-nasal and 94 (35%) in trans-oral settings. Although significantly more trans-nasal compared to trans-oral samples were culture-positive for S. pneumoniae at the primary diagnostic step (p<0.001) the opposite was observed in qPCR results (p<0.001). To confirm the presence of live pneumococcus in samples positive by qPCR but negative at the initial diagnostic step, we serially-diluted cell harvests, re-cultured and carefully examined for S. pneumoniae presence. Live pneumococci were recovered from an additional 43 parents including 42 positive in trans-oral and 4 in trans-nasal samples increasing the number of individuals culture- and qPCR-positive to 93 (35%) and positive by either of two methods to 107 (40%). There were significantly more trans-oral than trans-nasal samples positive for pneumococcus by both culture and qPCR (n = 71; 27%; vs. n = 50; 19%; p<0.05). Our data suggest that pneumococcal colonization is more common in adults than previously estimated and point towards the superiority of a trans-oral over a trans-nasal approach when testing adults for colonization with S. pneumoniae.