In vivo inhibition of thromboxane synthetase in infarcted canine myocardium

The in vivo effectiveness of the thromboxane synthetase inhibitor OKY-1581 was tested in normal and infarcted canine myocardium. A rapid in vitro assay was developed which permits an accurate assessment of the status of the tissue thromboxane synthetase at the time of sacrifice. Reperfused infarcts were created by two hours of coronary artery occlusion followed by release of occlusion and three days of recovery. OKY-1581 was infused at 100 micrograms/kg/min for 15 minutes, a dose previously found to cause an 85% inhibition of canine platelet thromboxane synthetase in vivo. The heart was rapidly excised and transmural tissue plugs of infarcted and normal areas were obtained. These were incubated for 5 minutes with prostaglandin endoperoxide (PGH2) in phosphate buffer. Thromboxane production was inhibited from 16 +/- 1 ng TxB2 per tissue plug to 5 +/- 1 in normal myocardium and from 27 +/- 5 to 6 +/- 1 in infarcted areas of myocardium. Control incubations showed no further inhibition with the in vitro addition of 20 micrograms/ml OKY-1581, confirming the completeness of in vivo inhibition. Thus significant inhibition of thromboxane synthetase by intravenous OKY-1581 occurs even in a reperfused zone of infarction.     

Sesquiterpenes of thalloid liverworts of the genera Conocephalum,Lunularia, Metzgeria and Riccardia

Thalloid liverworts of orders Metzgeriales and Marchantiales elaborate essential oils distinguishable from those of the Jungermanniales by the absence of β-barbatene and anastreptene. Riccardia sinuata elaborates a novel tricyclic exomethylene sesquiterpene of as yet undetermined structure. Conocephalum conicum elaborates cadinene-type sesquiterpenes. β-Cadinene from the latter species is clearly enantiomeric to the same product from vascular plants.     

Directed mutagenesis of DNA cloned in filamentous phage: influence of hemimethylated GATC sites on marker recovery from restriction fragments.

Gapped duplex DNA molecules of recombinant genomes of filamentous phage are constructed in vitro. Denatured restriction fragments covering (part of) the precisely constructed gap are hybridized to the gapped duplex DNA molecules to form ternary duplices. The two strands of the ternary duplex molecules carry different genetic markers within the region spanned by the restriction fragment leading to a one base pair mismatch or to an insertion loop of 93 nucleotides, respectively. The two strands also vary with respect to A-methylation in GATC sites. In cases of asymmetrical methylation, transfection of E. coli with these heteroduplex molecules leads to marker recoveries with a pronounced bias in favour of the marker encoded by the methylated strand. This effect at least partly explains the comparably low marker yields achieved in previous directed mutagenesis experiments using filamentous phage as the vector. The results suggest how these procedures can be optimized. Precise construction of a 93 bp insertion of 9.5% marker yield is described.     

Comparative studies of the structure and composition of the plasmalemma and the tonoplast in Saccharomyces cerevisiae

The two membranes, plasmalemma and tonoplast (Saccharomyces cerevisiae H 1022), are characterized ultrastructurally by their different texture in the corresponding freeze-fracture faces and their silver staining properties.Biochemical characterization with regard to proteins and lipids indicated that the ratio of protein to lipid is significantly higher in the plasmalemma as compared to the tonoplast. Moreover, a pronounced difference appears to exist for both the amount and the composition of total lipids, phospholipids and sterols. The protein patterns of the plasmalemma and the tonoplast reveal only minor differences, as judged by sodium dodecyl sulphate gel electrophoresis.     

Cultivation of Mesophilic Soil Crenarchaeotes in Enrichment Cultures from Plant Roots

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.     

Evolutionary transition pathways for changing peptide ligand specificity and structure

We identified evolutionary pathways for the inter- conversion of three sequentially and structurally unrelated peptides, GATPEDLNQKL, GLYEWGGARI and FDKEWNLIEQN, binding to the same site of the hypervariable region of the anti-p24 (HIV-1) monoclonal antibody CB4-1. Conversion of these peptides into each other could be achieved in nine or 10 single amino acid substitution steps without loss of antibody binding. Such pathways were identified by analyzing all 7 620 480 pathways connecting 2560 different peptides, and testing them for CB4-1 binding. The binding modes of intermediate peptides of selected optimal pathways were characterized using complete sets of substitution analogs, revealing that a number of sequential substitutions accumulated without changing the pattern of key interacting residues. At a distinct step, however, one single amino acid exchange induces a sudden change in the binding mode, indicating a flip in specificity and conformation. Our data represent a model of how different specificities, structures and functions might evolve in protein-protein recognition.     

Sequencing of pools of nucleic acids on oligonucleotide arrays

In sequencing-by-hybridization methods, the nucleotide sequence of a nucleic acid is reconstructed by overlapping oligonucleotides capable of hybridizing with the nucleic acid. In their present form, the methods are hardly suitable for sequencing of long nucleic acid molecules because of the occurrence of non-unique overlaps between the oligonucleotides, and similarly to the conventional sequencing methods, it is necessary to obtain an individual molecule. In the method described here, most ambiguities in reconstruction of a sequence from the constituent oligonucleotides are eliminated by preparing on oligonucleotide arrays and separate surveying of the nucleic acid nested partials. This enables longer nucleic acids to be sequenced, and results in a high redundancy of the input data allowing most hybridization errors to be eliminated by algorithmic means. Furthermore, large pools of nucleic acid strands can be sequenced directly, without isolating individual strands.