Hydrolysis of guanosine and adenosine 3',5'-monophosphates by rat blood.

Cyclic nucleotide phosphodiesterase activity was measured in whole blood, plasma, and suspensions of platelets and erythrocytes from rats. In fresh whole blood, apparent phosphodiesterase activity was low, but it rose strikingly during the hour after blood withdrawal. The apparent phosphodiesterase activity in platelet-free plasma showed no such increase, but that in platelet-enriched plasma increased in parallel with that in whole blood. The apparent phosphodiesterase activity of blood or of platelet-enriched plasma also was increased markedly by sonication. The increase in rat blood phosphodiesterase activity with aging thus appeared to be due to damage of platelets. Most of the phosphodiesterase activity in rat erythrocytes and platelets was located in the soluble fraction of sonicated preparations, but the total enzyme activities from the two sources exhibited marked differences in substrate specificity. With erythrocyte preparations, the rate of hydrolysis of muM concentrations of cyclic AMP was approx. 50 times that of cyclic GMP, while with platelet preparations, cyclic GMP was hydrolyzed about 20 times faster than cyclic AMP at muM levels. The activity of phosphodiesterase in platelets was much greater than that in erythrocytes at all concentrations of both substrates.     

Molecular phylogeny and a chronology of diversification for "phractocephaline" catfishes (Siluriformes: Pimelodidae) based on mitochondrial DNA and nuclear recombination activating gene 2 sequences

The Maracaibo basin in northwestern Venezuela is home to over 108 species of freshwater fishes, over half of which occur nowhere else. The rise of the Mérida Andes between 8 and 10 million years ago is believed to have divided a preexisting biota and facilitated allopatric speciation. The distinctive "phractocephaline" clade of pimelodid catfishes has a distribution that includes the Maracaibo historically and today is represented by Perrunichthys (Maracaibo endemic), Leiarius, Phractocephalus, and Steindachneridion. A resolved and well-supported phylogeny was obtained from the phylogenetic analysis of over 3.4kb (including cytochrome b, NADH dehydrogenase subunit 1, recombination activating gene subunit 2). Rates of divergence among "phractocephalines" were calibrated with fossil material and the Mérida uplift. These independent calibrators provided different node-age estimates when the rates were applied separately to the gene partitions (mtDNA and rag2). Node-age discrepancies were particularly evident at older nodes. This is due to two factors: (1) multiple substitution of mtDNA underestimates the amount of change and therefore time since cladogenesis, whereas (2) calibration of an ancient node with fossils produces an artificially slow rate (due to the masking of divergence through multiple substitution) that overestimates time when applied to younger nodes. Node-age estimates provided by the more slowly evolving rag2 sequences were more consistent with other sources of historical inference, e.g., paleogeography and the fossil record. Given these points, we provide a synthetic chronology of diversification and discuss the reasons for its preference. The phylogeny and synthetic chronology suggest Perrunichthys perruno (Maracaibo endemic) and Leiarius pictus (Orinoco and Guianas) to be a vicariant species pair that last shared a common ancestor during the period of Mérida uplift.     

Comprehensive Analysis of the Green-to-Blue Photoconversion of Full-Length Cyanobacteriochrome Tlr0924

Cyanobacteriochromes are members of the phytochrome superfamily of photoreceptors and are of central importance in biological light-activated signaling mechanisms. These photoreceptors are known to reversibly convert between two states in a photoinitiated process that involves a basic E/Z isomerization of the bilin chromophore and, in certain cases, the breakage of a thioether linkage to a conserved cysteine residue in the bulk protein structure. The exact details and timescales of the reactions involved in these photoconversions have not been conclusively shown. The cyanobacteriochrome Tlr0924 contains phycocyanobilin and phycoviolobilin chromophores, both of which photoconvert between two species: blue-absorbing and green-absorbing, and blue-absorbing and red-absorbing, respectively. Here, we followed the complete green-to-blue photoconversion process of the phycoviolobilin chromophore in the full-length form of Tlr0924 over timescales ranging from femtoseconds to seconds. Using a combination of time-resolved visible and mid-infrared transient absorption spectroscopy and cryotrapping techniques, we showed that after photoisomerization, which occurs with a lifetime of 3.6 ps, the phycoviolobilin twists or distorts slightly with a lifetime of 5.3 μs. The final step, the formation of the thioether linkage with the protein, occurs with a lifetime of 23.6 ms.     

Large Plasmids from Soil Bacteria Enriched on Halogenated Alkanoic Acids

Four Pseudomonas species and two Alcaligenes species were isolated from soil with a capacity to grow on halogenated alkanoic acids. They were shown to contain one of five large plasmids. The plasmids had molecular weights ranging from 98,800 to 190,000. They were associated with the ability to utilize the halogenated substrates 2-monochloropropionic acid and 2-monochloroacetic acid and with resistance towards one or more of the heavy metals mercury, selenium, and tellurium. The largest plasmid, pUU204, was shown to be unstable in continuous-flow culture when the organism was supplied with succinate as the sole carbon source. The dehalogenase gene associated with pUU204 appeared to be readily transferred to an incP group plasmid, R68-45.     

Methylation of nuclear DNA in Physarum polycephalum.

The restriction endonucleases HpaII and HhaI, whose action is inhibited by the presence of methylated base analogues at the recognition sequences in the DNA substrate, were used to investigate the distribution of 5-methylcytosine in nuclear DNA from Physarum polycephalum. Physarum DNA is digested into two fractions by these enzymes: a low-molecular-weight (M--) compartment comprising 80% of the DNA, and a high-molecular-weight (M+) compartment containing 20% of the DNA. The DNA fraction showing resistance to digestion by restriction endonuclease HpaII is cleaved by its isoschizomer MspI, indicating that methylated endonuclease-HpaII-specific sites are present in M + DNA. Additional properties of sequences in the M+ compartment were investigated.     

Replication of the deoxyribonucleic acid of multiple-drug-resistance factor in Escherichia coli.

1. It was shown that a system previously described for labelling R-factor DNA during transfer to an irradiated recipient strain of Escherichia coli did not allow high selectivity in the incorporation of thymine into R-factor DNA. 2. Lack of selectivity was shown to be due to cross-feeding from recipient to donor strain. 3. An improved system using a nalidixic acid-resistant recipient strain is described in which incorporation of thymine into the DNA of donor cells is minimized by addition of nalidixic acid after completion of transfer of the plasmid during conjugation.     

Steady-state and time-resolved spectroscopic studies on the hematoporphyrin-lipoprotein complex

The interaction of hematoporphyrin (Hp) with the isolated rabbit lipoprotein fractions very low density lipoproteins, low-density lipoproteins, and high-density lipoproteins has been studied by steady-state and time-resolved spectroscopy. The porphyrin appears to be bound to both the apoprotein and the lipid phase. The two populations of lipoprotein-bound Hp molecules can be distinguished on the basis of the fluorescence excitation spectrum, decay constants of the lowest excited singlet and triplet states, and accessibility to oxygen. Upon Hp binding, the intrinsic fluorescence emission of apolipoproteins is quenched at least in part via singlet-singlet energy transfer from tryptophyl residues to the porphyrin moiety. The binding of Hp with the protein matrix can be adequately described on the basis of Scatchard analysis, whereas the interaction of Hp with the lipid core can be described as the partitioning of the dye between a hydrophobic and an aqueous phase. The Hp binding capacity of lipoproteins is maximal for very low density lipoproteins.