Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.     

PCR Detection of Coxiella burnetii from Different Clinical Specimens, Especially Bovine Milk, on the Basis of DNA Preparation with a Silica Matrix

For PCR detection of Coxiella burnetii in various clinical specimens we developed a sample preparation method in which silica binding of DNA was used. This method was found to be fast, easily performed with large numbers of samples, and equally sensitive for all of the specimens tested (livers, spleens, placentas, heart valves, milk, blood). The DNA preparation method described here can also be used as an initial step in any PCR-based examination of specimens. The procedure was tested with more than 600 milk samples, which were taken from 21 cows that were seropositive for C. burnetii and reportedly had fertility problems (and therefore were suspected of shedding the agent through milk intermittently or continuously). Of the 21 cows tested, 6 were shedding C. burnetii through milk. Altogether, C. burnetii DNA was detected in 6% of the samples. There was no correlation between the shedding pattern and the serological results.     

Reconstitution of Mammary Gland Development In Vitro: Requirement of c-met and c-erbB2 Signaling for Branching and Alveolar Morphogenesis

We have established a cell culture system that reproduces morphogenic processes in the developing mammary gland. EpH4 mouse mammary epithelial cells cultured in matrigel form branched tubules in the presence of hepatocyte growth factor/scatter factor (HGF/SF), the ligand of the c-met tyrosine kinase receptor. In contrast, alveolar structures are formed in the presence of neuregulin, a ligand of c-erbB tyrosine kinase receptors. These distinct morphogenic responses can also be observed with selected human mammary carcinoma tissue in explant culture. HGF/SF-induced branching was abrogated by the PI3 kinase inhibitors wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. In contrast, neuregulin- induced alveolar morphogenesis was inhibited by the MAPK kinase inhibitor PD98059. The c-met–mediated response could also be evoked by transfection of a c-met specific substrate, Gab1, which can activate the PI3 kinase pathway. An activated hybrid receptor that contained the intracellular domain of c-erbB2 receptor suffices to induce alveolar morphogenesis, and was observed in the presence of tyrosine residues Y1028, Y1144, Y1201, and Y1226/27 in the substrate-binding domain of c-erbB2. Our data demonstrate that c-met and c-erbB2 signaling elicit distinct morphogenic programs in mammary epithelial cells: formation of branched tubules relies on a pathway involving PI3 kinase, whereas alveolar morphogenesis requires MAPK kinase.     

A new Hyphoderma from Europe

The new species Hyphoderma tibia (Aphyllophorales, Basidiomycotina) is described and illustrated using drawings and scanning electron microscope.     

Tolerance of the resting cysts of Colpoda inflata (Ciliophora,Colpodea) and Meseres corlissi (Ciliophora,Spirotrichea) to desiccation and freezing

The survival of ciliate resting cysts, in the presence and absence of soil, was studied under two environmental stresses: desiccation and freezing. Laboratory strains of the common species Colpoda inflata and the rare species Meseres corlissi were used in these experiments, which yielded the following results: 1) Freezing of cysts in soil with a residual moisture level exceeding ～30% was destructive for both species. 2) Survival of Meseres corlissi cysts depended largely on the presence of soil. 3) In the absence of soil, Colpoda inflata cysts had greater tolerance to desiccation and freezing than Meseres corlissi cysts. Possible consequences for the distribution of natural populations are discussed.     

Interaction of heparin with cationic molecular probes: Probe charge is a major determinant of binding stoichiometry and affinity

Fluorescent perylenediimide probes modified with 2, 4, 6, or 8 ammonium groups were synthesized and their binding to the antithrombotic drug heparin was studied by fluorescence spectroscopy in solution. The polyanionic polysaccharide strands of heparin bind more probe molecules per sugar unit when the charge of the latter is low, and stability of the probe-heparin complex increases with increasing probe charge.     

Timing of reproduction in a Darwin''s finch: temporal opportunism under spatial constraints

We investigated whether predation by the minor grison ( Galictis cuja , a small mustelid) played a key role in limiting a wild cavy population ( Cavia magna ), ultimately leading to its local extinction. Radio-telemetry and capture-mark-recapture techniques were used to estimate grison predation rates (kill rates), time-specific probabilities of apparent mortality (population loss rate), overall mortality and grison predation for the cavy population. Additionally, we present data on alternative prey species, grison diet and reproduction to show potential proximate mechanisms of grison predation on wild cavies. The predictions specified were mostly confirmed: (1) grison predation was responsible for almost 80% of the cavies killed by known predators; (2) grison predation probabilities paralleled those of overall mortality of cavies over time; and (3) also those of the apparent mortality of the population. Thus, the population dynamics and the local extinction of the cavy population were not due to emigration processes. (4) Grison predation rates were not density-dependent, but showed pronounced peaks during the austral summer. The grison mainly preyed on small mammals: two water-rat species and the wild cavies. When the availability of alternative prey decreased in summer, the grison appeared to specialise on cavies. The onset of grison reproduction was somewhat delayed in relation to the onset of cavy reproduction. The lack of alternative prey coincided with high grison food demands due to reproduction, leading to a very high predation pressure ultimately resulting in the local extinction of the cavy population. We conclude that grison predation was indeed the main factor driving changes of the cavy population studied and speculate why caviomorph rodents might be especially susceptible to local extinction processes.     

Variants in Nebulin (NEB) Are Linked to the Development of Familial Primary Angle Closure Glaucoma in Basset Hounds

Several dog breeds are susceptible to developing primary angle closure glaucoma (PACG), which suggests a genetic basis for the disease. We have identified a four-generation Basset Hound pedigree with characteristic autosomal recessive PACG that closely recapitulates PACG in humans. Our aim is to utilize gene mapping and whole exome sequencing approaches to identify PACG-causing sequence variants in the Basset. Extensive clinical phenotyping of all pedigree members was conducted. SNP-chip genotyping was carried out in 9 affected and 15 unaffected pedigree members. Two-point and multipoint linkage analyses of genome-wide SNP data were performed using Superlink-Online SNP-1.1 and a locus was mapped to chromosome 19q with a maximum LOD score of 3.24. The locus contains 12 Ensemble predicted canine genes and is syntenic to a region on chromosome 2 in the human genome. Using exome-sequencing analysis, a possibly damaging, non-synonymous variant in the gene Nebulin (NEB) was found to segregate with PACG which alters a phylogenetically conserved Lysine residue. The association of this variants with PACG was confirmed in a secondary cohort of unrelated Basset Hounds (p = 3.4 × 10^-4, OR = 15.3 for homozygosity). Nebulin, a protein that promotes the contractile function of sarcomeres, was found to be prominently expressed in the ciliary muscles of the anterior segment. Our findings may provide insight into the molecular mechanisms that underlie PACG. The phenotypic similarities of disease presentation in dogs and humans may enable the translation of findings made in this study to patients with PACG.