PubMed · 15412346
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H L PEARCE. 1950. Custom base shoes.. https://pubmed.ncbi.nlm.nih.gov/15412346/
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We study in HMC-1 the activation process, measured as histamine release. We know that ammonium chloride (NH(4)Cl) and ionomycin release histamine, and the modulatory role of drugs targeting protein kinase C (PKC), adenosine 3',5'-cyclic monophosphate (cAMP), tyrosine kinase (TyrK) and phosphatidylinositol 3-kinase (PI3K) on this effect. We used Gö6976 (100 nM) and low concentration of GF 109203X (GF) (50 nM) to inhibit Ca(2+)-dependent PKC isozymes. For Ca(2+)-independent isozymes, we used 500 nM GF and 10 microM rottlerin (specifically inhibits PKCdelta). Phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) was used to stimulate PKC, and genistein (10 microM) and lavendustin A (1 microM) as unspecific TyrK inhibitors. STI571 10 microM was used to specifically inhibit the activity of Kit, the receptor for stem cell factor, and 10 nM wortmannin as a PI3K inhibitor. Activation of PKC with PMA enhances histamine release in response to NH(4)Cl and ionomycin. PMA increases NH(4)Cl-induced alkalinization and ionomycin-induced Ca(2+) entry. Inhibition of PKCdelta strongly inhibits Ca(2+) entry elicited by ionomycin, but failed to modify histamine release. The effect of cAMP-active drugs was explored with the adenylate cyclase activator forskolin (30 microM), the inhibitor SQ22,536 (1 microM), the cAMP analog dibutyryl cAMP (200 microM), and the PKA blocker H89 (1 microM). Forskolin and dibutyryl cAMP do increase NH(4)Cl-induced alkalinization, and potentiate histamine release elicited by this compound. Our data indicates that alkaline-induced exocytosis is modulated by PKC and cAMP, suggesting that pH could be a modulatory signal itself.
The present study describes the improvement of a technique, the alkaline-halo assay (AHA), for the assessment of DNA single-strand breakage at the single-cell level. AHA involves a series of sequential steps in which cells are embedded in melted agarose and spread onto microscope slides, incubated in a high-salt alkaline lysis solution, then in a hypotonic alkaline solution and, finally, stained with ethidium bromide (EB). Under these conditions, single-stranded DNA fragments diffuse radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nuclear remnants: the area of the halo is a direct function of the extent of DNA strand scission. These phenomena can be conveniently monitored with a fluorescence microscope and quantified by image-processing analysis. The behaviour of single-stranded DNA fragments under the conditions of the modified assay, called fast halo assay (FHA), is essentially the same as in AHA. The modifications consist in the simplification of the lysis, denaturation and staining procedures, and allow, as compared with AHA, the preparation of samples within 15 min, with a two-third reduction in total processing time, using only two reagents to promote DNA extraction and staining: NaOH and EB. A variation of the FHA operating at non-denaturing conditions to discriminate apoptotic cells from non-apoptotic cells bearing DNA single-strand breaks is also illustrated. To benchmark FHA sensitivity and reliability, the DNA single-strand breaks (SSBs) resulting either from exposure of cultured mammalian cells to different DNA-damaging agents or from secondary apoptotic DNA cleavage, have been quantified and results compared with the outcomes of reference techniques run in parallel, namely AHA, comet assay and Hoechst 33342 staining. The results indicate that FHA has the same reliability and sensitivity of the reference assays, but presents the additional advantages of being inexpensive, more rapid and strikingly simple.
Bacterial diversity in alkaline (pH 10.4) and permanently cold (4 degrees C) ikaite tufa columns from the Ikka Fjord, SW Greenland, was investigated using growth characterization of cultured bacterial isolates with Terminal-restriction fragment length polymorphism (T-RFLP) and sequence analysis of bacterial 16S rRNA gene fragments. More than 200 bacterial isolates were characterized with respect to pH and temperature tolerance, and it was shown that the majority were cold-active alkaliphiles. T-RFLP analysis revealed distinct bacterial communities in different fractions of three ikaite columns, and, along with sequence analysis, it showed the presence of rich and diverse bacterial communities. Rarefaction analysis showed that the 109 sequenced clones in the 16S rRNA gene library represented between 25 and 65% of the predicted species richness in the three ikaite columns investigated. Phylogenetic analysis of the 16S rRNA gene sequences revealed many sequences with similarity to alkaliphilic or psychrophilic bacteria, and showed that 33% of the cloned sequences and 33% of the cultured bacteria showed less than 97% sequence identity to known sequences in databases, and may therefore represent yet unknown species.