3-Carboxy-6-chloro-7-hydroxycoumarin: A highly fluorescent, water-soluble violet-excitable dye for cell analysis

In our search for new violet-excitable dyes with improved photophysical and photochemical properties, we examined several halogen-substituted hydroxycoumarins and found that chlorinated derivatives are at least as bright as their fluorinated analogs. A monochlorinated hydroxycoumarin was found to have a high quantum yield (∼0.98), and human leucocyte-specific monoclonal antibodies (CD3, CD4, and CD45) conjugated with this dye exhibited reliable performance in flow cytometry assays. Additional studies were performed, with BD Horizon V450-antibody conjugates being included in eight-color cocktails aimed at subsetting lymphocytes and myeloid cells. Such cocktails can frequently be unstable due to the tendency of one or more components to lose structural integrity, photobleach, or develop unwanted affinities for another component. However, the cocktails employed in this study enabled several different applications to be run and established that multicolor reagent mixtures containing V450-antibody conjugates are functional and stable.     

Filograna (colonial serpulid worm tubes) from Upper Triassic (Norian) reef boulders of Taurus Mts. (southern Turkey)

Two colonial serpulid worm tubes—Filograna taurica n. sp. and Filograna serialis n. sp.—from the Upper Triassic (Norian) reef boulders occurring in Taurus Mts. (south Turkey) are described. As bafflers, both species of Filograna build microbioherms reaching dimensions of up to 15 cm. Both species were not known either in Turkey or in any other Triassic localities of the Tethyan realm.     

Preparation and properties of three specific active derivatives of ribonuclease A obtained by methylation of methionine residues in 8 M urea.

In 8 M urea at low pH, CH3I reacts specifically with the four methionine residues of ribonuclease A, and all four residues react at the same rate. Uon removal of the denaturant, only unmodified ribonuclease and 3 of the 15 possible derivatives modified on methionine refold to regenerate activity. All the enzymatic activity is recored after chromatography on IRC-50 and the four active proteins separate from each other and from the 12 inactive derivatives, which are not eluted from the resin under the conditions used. By the use of 14CH3I, performic acid oxidation, chymotryptic digestion, and separation of the resulting peptides by ion exchange, the active species were determined to be unmodified ribonuclease, CH3Met-29-RNase, CH3Met-79-RNase, and CH3Met-29, CH3Met-79-RNase. these proteins have melting temperatures of 63, 58, 43, and 36 degrees, respectively, at pH 6.3-70. Methylation at methionine-29 or -79 has no effect on enzymatic activity. Conversely, methylation at methionine-13 or -30 prevents refolding to an active conformation at 25 degrees elution from IRC-50. These results are consistent with the positions of the four methionine residues in crystals of ribonuclease A and ribonuclease S as determined by X-ray diffraction.     

The <Emphasis Type="Italic">disarrayed</Emphasis> mutation results in cell cycle and neurogenesis defects during retinal development in zebrafish

Background  

The vertebrate retina is derived from proliferative neuroepithelial cells of the optic cup. During retinal development, cell proliferation and the processes of cell cycle exit and neurogenesis are coordinated in neuroepithelial progenitor cells. Previous studies have demonstrated reciprocal influences between the cell cycle and neurogenesis. However the specific mechanisms and exact relationships of cell cycle regulation and neurogenesis in the vertebrate retina remain largely unknown.     

Visualization of isozymes which generate inorganic phosphate

A sensitive, new enzymatic method for the detection of isozymes which liberate inorganic phosphate (or pyrophosphate) is described. The new method for the detection of phosphate differs from the established method for nucleoside phosphorylase by the substitution of one reagent. The new enzymatic method, compared to existing methods for the detection of phosphate on electrophoretic gels, is advantageous due to its sensitivity and generation of a nondiffusible formazan chromogenic product.     

Leakage of adenylates during cold methanol/glycerol quenching of Escherichia coli

Effective and rapid inactivation of cellular metabolism is a prerequisite for accurate metabolome analysis. Cold methanol quenching is commonly applied to stop any metabolic activity and, at the same time remaining the cells’ integrity. However, it is reported that especially prokaryotic cells like Escherichia coli and Corynebacterium glutamicum tend to leak intracellular metabolites during cold methanol quenching. In this work leakage of adenylates is quantified for different quenching fluids. Further, a methanol/glycerol based quenching fluid is proposed, which reduces leakage drastically compared to the commonly applied methanol/water solution (16% ATP leakage compared to more than 70%).