On the influence of oxygen and cell concentration in an SFPR whole cell biocatalytic Baeyer-Villiger oxidation process

Efficient whole cell biotransformations, in particular microbial whole cell Baeyer-Villiger oxidation with molecular oxygen, demand comprehension and optimization of the process details involved. Optimal provision of oxygen and control of bioprocess parameters are pivotal for their success. The interrelation of cell density and oxygen supply in an in situ substrate feeding and product removal (SFPR) whole cell Baeyer-Villiger oxidation process was investigated in detail. Both parameters were optimized with respect to practical considerations. The outcome of this study supports a schematic process model, allows estimation of optimum process conditions and exploration of its limits.     

Isolation and characterization of plaque-purified strains of Malacosoma disstria Nucleopolyhedrovirus

Seven plaque-purified genotypic variants or strains, derived from a previously described field isolate of the Malacosoma disstria Nucleopolyhedrovirus (MadiNPV) from Alberta populations of forest tent caterpillar, were characterized based on distinctive restriction endonuclease fragment patterns. Two strains, MadiNPV-pp3 and MadiNPV-pp11, were selected for further characterization, as they represented strains producing high and low budded virus (BV) titres, respectively, in the M. disstria cell line UA-Md203. Analysis of restriction endonuclease fragment profiles indicated the genomes differed significantly in size, 133.8 +/- 2.4 kb for MadiNPV-pp3 and 118.1 +/- 3.5 kb for MadiNPV-pp11. These strains were characterized based on their BV production in three different cell lines derived from M. disstria haemocytes. Compared with MadiNPV-pp11, MadiNPV-pp3 produced two- to three-fold more BVs in UA-Md203 and 210 other cell lines; however, BV production was only marginally higher for MadiNPV-pp3 in the UA-Md221 cell line. Similarly, the yield of polyhedral inclusion bodies was significantly higher for MadiNPV-pp3 in UA-Md203 and 210 cell lines than for MadiNPV-pp11 but not in the UA-Md221 cell line. This data, although derived from a limited number of cell lines, suggested MadiNPV-pp3 may have a broader tissue tropism than MadiNPV-pp11.     

Species divergence and relationships in Stephanomeria (Compositae): PgiC phylogeny compared to prior biosystematic studies

We present a maximum likelihood tree of 41 PgiC sequences for the monophyletic Stephanomeria, with 10 perennial and six annual species, widely distributed in western North America and exemplary of different speciation processes. The phylogenetic analysis represents the first use of PgiC sequences for Compositae. The annual species were originally delimited by biosystematic studies that provided evidence of their reproductive compatibility and chromosome structural homology. The perennial species are highly distinctive in morphology and have not been examined similarly. The PgiC tree provides more resolution than our previous ITS/ETS tree and reflects both past and ongoing hybridization and/or incomplete lineage sorting. Two major PgiC clades were resolved in Stephanomeria. One clade contains the genes from the annual species plus the perennial, insular endemic S. guadalupensis, which appears closely related to a monophyletic S. virgata. Stephanomeria exigua is not monophyletic. The second clade includes the genes from all other sampled perennial species and a monophyletic subclade of four genes from two annual species. The results are compared to previous studies, also using PgiC, of Clarkia (Onagraceae). Both molecular systematic and biosystematic approaches are essential to discern the very different courses of evolution in these two, well-studied genera of western North America.     

Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity

HS (heparan sulfate) is essential for normal embryonic development. This requirement is due to the obligatory role for HS in the signalling pathways of many growth factors and morphogens that bind to sulfated domains in the HS polymer chain. The sulfation patterning of HS is determined by a complex interplay of Golgi-located N- and O-sulfotransferases which sulfate the heparan precursor and cell surface endosulfatases that selectively remove 6-O-sulfates from mature HS chains. In the present study we generated single or double knock-out mice for the two murine endosulfatases mSulf1 and mSulf2. Detailed structural analysis of HS from mSulf1-/- fibroblasts showed a striking increase in 6-O-sulfation, which was not seen in mSulf2-/- HS. Intriguingly, the level of 6-O-sulfation in the double mSulf1-/-/2-/- HS was significantly higher than that observed in the mSulf1-/- counterpart. These data imply that mSulf1 and mSulf2 are functionally co-operative. Unlike their avian orthologues, mammalian Sulf activities are not restricted to the highly sulfated S-domains of HS. Mitogenesis assays with FGF2 (fibroblast growth factor 2) revealed that Sulf activity decreases the activating potential of newly-synthesized HS, suggesting an important role for these enzymes in cell growth regulation in embryonic and adult tissues.     

The pathogen-host interactions database (PHI-base) provides insights into generic and novel themes of pathogenicity

Fungal and oomycete pathogens of plants and animals are a major global problem. In the last 15 years, many genes required for pathogenesis have been determined for over 50 different species. Other studies have characterized effector genes (previously termed avirulence genes) required to activate host responses. By studying these types of pathogen genes, novel targets for control can be revealed. In this report, we describe the Pathogen-Host Interactions database (PHI-base), which systematically compiles such pathogenicity genes involved in pathogen-host interactions. Here, we focus on the biology that underlies this computational resource: the nature of pathogen-host interactions, the experimental methods that exist for the characterization of such pathogen-host interactions as well as the available computational resources. Based on the data, we review and analyze the specific functions of pathogenicity genes, the host-specific nature of pathogenicity and virulence genes, and the generic mechanisms of effectors that trigger plant responses. We further discuss the utilization of PHI-base for the computational identification of pathogenicity genes through comparative genomics. In this context, the importance of standardizing pathogenicity assays as well as integrating databases to aid comparative genomics is discussed.     

Phylogeny of Cephalobina (Nematoda): molecular evidence for recurrent evolution of probolae and incongruence with traditional classifications

Nematodes of the suborder Cephalobina include an ecologically and morphologically diverse array of species that range from soil-dwelling microbivores to parasites of vertebrates and invertebrates. Despite a long history of study, certain of these microbivores (Cephaloboidea) present some of the most intractable problems in nematode systematics; the lack of an evolutionary framework for these taxa has prevented the identification of natural groups and inhibited understanding of soil biodiversity and nematode ecology. Phylogenetic analyses of ribosomal (LSU) sequence data from 53 taxa revealed strong support for monophyly of taxa representing the Cephaloboidea, but do not support the monophyly of most genera within this superfamily. Historically these genera have primarily been recognized based on variation in labial morphology, but molecular phylogenies show the same general labial (probolae) morphotype often results from recurrent similarity, a result consistent with the phenotypic plasticity of probolae previously observed for some species in ecological time. Phylogenetic analyses of LSU rDNA also recovered strong support for some other groups of cephalobs, including taxa representing most (but not all) Panagrolaimoidea. In addition to revealing homoplasy of probolae, molecular trees also imply other unexpected patterns of character evolution or polarity, including recurrent similarity of offset spermatheca presence, and representation of complex probolae as the ancestral condition within Cephaloboidea. For Cephalobidae, molecular trees do not support traditional genera as natural groups, but it remains untested if deconstructing probolae morphotypes or other structural features into finer component characters may reveal homologies that help delimit evolutionary lineages.     

The most C-terminal tri-glycine segment within the polyglycine stretch of the pea Toc75 transit peptide plays a critical role for targeting the protein to the chloroplast outer envelope membrane

The protein translocation channel at the outer envelope membrane of chloroplasts (Toc75) is synthesized as a larger precursor with an N-terminal transit peptide. Within the transit peptide of the pea Toc75, a major portion of the 10 amino acid long stretch that contains nine glycine residues was shown to be necessary for directing the protein to the chloroplast outer membrane in vitro. In order to get insights into the mechanism by which the polyglycine stretch mediates correct targeting, we divided it into three tri-glycine segments and examined the importance of each domain in targeting specificity in vitro. Replacement of the most C-terminal segment with alanine residues resulted in mistargeting the protein to the stroma, while exchange of either of the other two tri-glycine regions had no effect on correct targeting. Furthermore, simultaneous replacement of the N-terminal and middle tri-glycine segments with alanine repeats did not cause mistargeting of the protein as much as those of the N- and C-terminal, or the middle and C-terminal segments. These results indicate that the most C-terminal tri-glycine segment is important for correct targeting. Exchanging this portion with a repeat of leucine or glutamic acid also caused missorting of Toc75 to the stroma. By contrast, its replacement with repeats of asparagine, aspartic acid, serine, and proline did not largely affect correct targeting. These data suggest that relatively compact and nonhydrophobic side chains in this particular region play a crucial role in correct sorting of Toc75.     

Synthesis and monoamine transporter affinity of 2beta-carbomethoxy-3beta-(4'-p-substituted phenyl)-piperidine analogs of cocaine

A series of novel piperidine based analogs of cocaine was synthesized and evaluated in vitro against the three monoamine transporters to develop new potential selective SERT radiotracers. Modification of the phenyl substitution with five-membered heterocyclic groups resulted in a wide affinity and selectivity scale. Radiolabeling and mouse in vivo study was performed on the piperidine analog of ZIENT, which crossed the blood-brain barrier but failed to selectively accumulate in the regions of the brain rich in SERT.     

Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm Formation in Response to Angiotensin II

Aortic aneurysm is dilation of the aorta primarily due to degradation of the aortic wall extracellular matrix (ECM). Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs), the proteases that degrade the ECM. Timp3 is the only ECM-bound Timp, and its levels are altered in the aorta from patients with abdominal aortic aneurysm (AAA). We investigated the causal role of Timp3 in AAA formation. Infusion of angiotensin II (Ang II) using micro-osmotic (Alzet) pumps in Timp3^−/− male mice, but not in wild type control mice, led to adverse remodeling of the abdominal aorta, reduced collagen and elastin proteins but not mRNA, and elevated proteolytic activities, suggesting excess protein degradation within 2 weeks that led to formation of AAA by 4 weeks. Intriguingly, despite early up-regulation of MMP2 in Timp3^−/−Ang II aortas, additional deletion of Mmp2 in these mice (Timp3^−/−/Mmp2^−/−) resulted in exacerbated AAA, compromised survival due to aortic rupture, and inflammation in the abdominal aorta. Reconstitution of WT bone marrow in Timp3^−/−/Mmp2^−/− mice reduced inflammation and prevented AAA in these animals following Ang II infusion. Treatment with a broad spectrum MMP inhibitor (PD166793) prevented the Ang II-induced AAA in Timp3^−/− and Timp3^−/−/Mmp2^−/− mice. Our study demonstrates that the regulatory function of TIMP3 is critical in preventing adverse vascular remodeling and AAA. Hence, replenishing TIMP3, a physiological inhibitor of a number of metalloproteinases, could serve as a therapeutic approach in limiting AAA development or expansion.