A simplified HPLC method for simultaneously quantifying ribonucleotides and deoxyribonucleotides in cell extracts or frozen tissues

Agents and conditions that induce alterations in deoxyribonucleotide pools can have important regulatory effects on the rate of DNA synthesis as well as cell cycle progression. A simplified procedure for the separation of both ribonucleoside triphos-phates (NTP) and deoxyribonucleoside triphosphates (dNTP) is presented which utilizes reversed phase high-performance liquid chromatography coupled with diode array detection. The simultaneous resolution of NTP and dNTP peaks within the same cell extract effectively eliminates the need for post-extraction steps such as periodate oxidation and/or boronate affinity chromatography previously used to degrade or isolate co-eluting NTP from dNTP. The resolution of two nucleotides, dGTP and ADP, was found empirically to vary with the efficiency of the C18 column. High efficiency columns (>90 000 plates/m) provided good separation; however, less efficient columns resulted in co-elution of dGTP and ADP. These co-eluting nucleotides can be accurately quantified, if necessary, using diode array technology and a mathematical expression which incorporates molar peak coefficients and peak areas obtained by monitoring at dual wavelengths. Tissue samples or single cell suspensions were extracted with trichloroacetic acid and the neutralized extract was injected directly into the column without prior lyophilization. The per cent recovery of standards was .99% and replicate extractions within or between samples were highly reproducible (sd<5%). The single step method described minimizes potential losses associated with post-extraction manipulation and provides the capability to examine alterations in nucleotide precursor–product metabolism under various physiological and pharmacological conditions.     

Identification of the Serratia endonuclease dimer: structural basis and implications for catalysis.

The Serratia endonuclease is an extracellularly secreted enzyme capable of cleaving both single- and double-stranded forms of DNA and RNA. It is the first member of a large class of related and usually dimeric endonucleases for which a structure is known. Using X-ray crystallography, the structure of monomer of this enzyme was reported by us previously (Miller MD et al., 1994, Nature Struct Biol 1:461-468). We now confirm the dimeric nature of this enzyme through light-scattering experiments and identify the physiologic dimer interface through crystal packing analysis. This dimerization occurs through an isologous twofold interaction localized to the carboxy-terminal subdomain of the enzyme. The dimer is a prolate ellipsoid with dimensions 30 A x 35 A x 90 A. The dimer interface is flat and contains four salt links, several hydrogen bonds, and nonpolar interactions. Buried water is prominent in this interface and it includes an unusual "cubic" water cluster. The position of the two active sites in the dimer suggests that they can act independently in their cleavage of DNA, but have a geometrical advantage in attacking substrate relative to the monomer.     

The effect of an interleukin receptor antagonist (IL-1ra) on colonocyte eicosanoid release

We investigated whether an interleukin 1 receptor antagonist (IL-1ra) altered cellular release of prostanoids and leukotrienes in a transformed colonic cell line (CACO-2) in the presence of proinflammatory stimuli. Cellular inflammation was induced by treatment with lipopolysaccharide (LPS) or the cytokine, interleukin 1 beta (IL-1(beta)). In a separate set of experiments, cells were pretreated with IL-1ra prior to exposure to LPS or IL-1(beta). Prostaglandin E(2) and leukotriene B(4) (LTB(4)) levels were quantified by ELISA assays. Both LPS and IL-1(beta) exposure were noted to stimulate cellular PGE(2) release, a response which was significantly inhibited by IL-1ra treatment. Either stimulant when administered alone failed to stimulate release of LTB(4). When administered after IL-1ra pretreatment however, both stimuli caused a significant increase in LTB(4) release. These results suggest that a cytokine receptor antagonist can selectively influence eicosanoid production in this cell line. Furthermore, this study suggests that a IL-1ra may have a future clinical role in the treatment of inflammatory disorders of the colon which are intimately linked to enhanced eicosanoid synthesis.     

Effect of subzero incubation on fluoride binding by laccase

We have refined a useful incubation method for preparing adducts of tree laccase with inhibitor anions; however, the technique should have wider application. The procedure involves incubating a previously frozen aqueous solution at subzero temperature. Factors influencing the amount of adduct that forms include the nature of the buffer, pH changes that occur at subzero temperatures and the presence of glycerol. In the absence of a glassing agent like glycerol, the phase separation and solute pooling that occur with ice formation help drive adduct formation. The results reveal several important factors to consider in designing or interpreting low-temperature spectroscopic investigations.     

Historical Analysis of Genetic Variation Reveals Low Effective Population Size in a Northern Pike (Esox Lucius) Population

Effective population size (N(e)) of a natural fish population was estimated from temporal changes in allele frequencies at seven microsatellite loci. Use of a historical collection of fish scales made it possible to increase the precision of estimates by increasing the time interval between samples and to use an equation developed for discrete generations without correcting for demographic parameters. Estimates of N(e) for the time intervals 1961-1977 and 1977-1993 were 35 and 72, respectively. For the entire interval, 1961-1993, the estimate of N(e) was 48 when based on a weighted mean derived from the above two estimates or 125 when calculated from 1961 and 1993 samples only. Corresponding ratios of effective size to adult census size ranged from 0.03 to 0.14. An N(e) of 48 over a 32-year period would imply that this population lost as much as 8% of its heterozygosity in that time. Results suggest the potential for using genetic methods based on microsatellite loci data to compare historical trends in N(e) with population dynamic parameters. Such comparisons will help to evaluate the relationship between genetic diversity and long-term persistence of natural populations.     

Morphotypes of friable embryogenic maize callus

Friable, embryogenic callus cultures of maize are normally maintained as a heterogeneous mixture of various morphotypes in different stages of development (Fransz and Schel 1991). With stringent selection during subculture, three Type II callus morphotypes have been enriched and maintained. Cultured tissue segments from the three morphotypes referred to as pre-embryogenic, early embryogenic, and late embryogenic were analyzed using light and scanning electron microscopy. The results suggest a developmental relationship between the three morphotypes. Regeneration studies substantiated that all morphotypes were capable of regenerating through somatic embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - MS Murashige and Skoog(1962) Medium - SEM Scanning Electron Microscopy - FAA Formalin-acetic acid-alcohol     

Carbon and energy yields in prebiotic syntheses using atmospheres containing CH4, CO and CO2

Yields based on carbon are usually reported in prebiotic experiments, while energy yields (moles cal^–1) are more useful in estimating the yields of products that would have been obtained from the primitive atmosphere of the earth. Energy yields for the synthesis of HCN and H₂CO from a spark discharge were determined for various mixtures of CH₄, CO, CO₂, H₂, H₂O, N₂ and NH₃. The maximum yields of HCN and H₂CO from CH₄, CO, and CO₂ as carbon sources are about 4×10^–8 moles cal^–1.