K-Opioid Agonist Modulation of [3H]Thymidine Incorporation into DNA: Evidence for the Involvement of Pertussis Toxin-Sensitive G Protein-Coupled Phosphoinositide Turnover

Abstract: A body of evidence has indicated that μ-opioid agonists can inhibit DNA synthesis in developing brain. We now report that K -selective opioid agonists (U69593 and U50488) modulate [³H]thymidine incorporation into DNA in fetal rat brain cell aggregates in a dose- and developmental stage-dependent manner. K agonists decreased thymidine incorporation by 35% in cultures grown for 7 days, and this process was reversed by the K -selective antagonist, norbinaltorphimine, whereas in 21-day brain cell aggregates a 3,5-fold increase was evident. Cell labeling by [³H]thymidine was also inhibited by the K -opioid agonist as shown by autoradiography. In addition, U69593 reduced basal rates of phosphoinositide formation in 7-day cultures and elevated it in 21-day cultures. Control levels were restored by norbin-altorphimine. Pertussis toxin blocked U69593-mediated inhibition of DNA synthesis. The action of K agonists on thymidine incorporation in the presence of chelerythrine, a protein kinase C (PKC) inhibitor, or in combination with LiCl, a noncompetitive inhibitor of inositol phosphatase, was attenuated in both 7- and 21-day cultures. These results suggest that K agonists may inhibit DNA synthesis via the phosphoinositide system with a pertussis toxin-sensitive G protein as transducer. In mixed glial cell aggregates, U50488 increased thymidine incorporation into DNA 3.1-fold, and this stimulation was reversed by the opioid antagonist naltrexone.     

Multiple binding sites for cellular proteins in the 3' end of Sindbis alphavirus minus-sense RNA.

The 3' end of Sindbis virus minus-sense RNA was tested for its ability to bind proteins in mosquito cell extracts, using labeled riboprobes that represented different parts of this region. We found four domains in the first 250 nucleotides that could bind the same 50- and 52-kDa proteins, three with high affinity and one with low affinity, whereas tested domains outside this region did not bind these proteins. The first binding domain was found in the first 60 nucleotides, which represents the complement of the 5'-nontranslated region, the second in the next 60 nucleotides, the third in the following 60 nucleotides, and the fourth between nucleotides 194 and 249 (all numbering is 3' to 5'). The relative binding constants, Kr, of the first, second, and fourth sites were similar, whereas that of domain 2 was fivefold less. Deletion mapping of the first domain showed that the first 10 nucleotides were critical for binding. Deletion of nucleotides 2 to 4, deletion or replacement of nucleotide 5, or deletion of the first 15 nucleotides was deleterious for binding, deletion of nucleotides 10 to 15, 26 to 40, or 41 to 55 had little effect on the binding, and deletion of nucleotides 15 to to 25 increased the binding affinity. We also found that the corresponding riboprobes derived from two other alphaviruses, Ross River virus and Semliki Forest virus, and from rubella virus were also able to interact with the 50- and 52-kDa proteins. The Kr value for the Semliki Forest virus probe was similar to that for the Sindbis virus probe, while that for the Ross River virus probe was four times greater. The rubella virus probe was bound only weakly, consistent with the fact that mosquito cells are not permissive for rubella virus replication. We suggest that the binding of the 50- and 52-kDa proteins to the 3' end of alphavirus minus-sense RNA represents an important step in the initiation of RNA replication.     

Chloroplast DNA Diversity among Trees, Populations and Species in the California Closed-Cone Pines (Pinus Radiata, Pinus Muricata and Pinus Attenuata)

The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% of the chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [G(st) = 84(+/-13)%]; nucleotide diversity among species was estimated to be 0.3(+/-0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [G(st) = 87(+/-8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/-0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga.     

Volatile sensation: The chemical ecology of the earthy odorant geosmin

Geosmin may be the most familiar volatile compound, as it lends the earthy smell to soil. The compound is a member of the largest family of natural products, the terpenoids. The broad distribution of geosmin among bacteria in both terrestrial and aquatic environments suggests that this compound has an important ecological function, for example, as a signal (attractant or repellent) or as a protective specialized metabolite against biotic and abiotic stresses. While geosmin is part of our everyday life, scientists still do not understand the exact biological function of this omnipresent natural product. This minireview summarizes the current general observations regarding geosmin in prokaryotes and introduces new insights into its biosynthesis and regulation, as well as its biological roles in terrestrial and aquatic environments.     

Nanodiamond-promoted white light emission in Eu,Dy, and Cu-containing phosphate glass: quantitative analysis and colorimetric study

A thorough analysis of glass containing Eu₂O₃ and Dy₂O₃, or Eu₂O₃, Dy₂O₃, and CuO melted together with nanodiamond powder was pursued based on measurements of optical absorption, photoluminescence (PL) emission and excitation spectra, and colorimetry. Nanodiamond facilitated the stabilization of Cu⁺ and Eu²⁺ ions with blue-emitting characteristics that, along with yellow-emitting Dy³⁺ and red-emitting Eu³⁺ led to the white light-emitting glass. Novel intensity notations implemented in intensity-based spectral ratios, and difference intensity correlation analysis were proposed for the assessment of PL properties. The chromaticity and correlated colour temperature of the emission were ultimately investigated as a two-parametric problem based on: (1) the different ionic components; and (2) the various excitation wavelengths employed. The optical analysis approach adds to the characterization methods to further fundamental understanding and provide helpful analytical tools for designing materials for tunable white light-emitting devices.