Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase

The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee >99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications.     

Imaging collagen in intact viable healthy and atherosclerotic arteries using fluorescently labeled CNA35 and two-photon laser scanning microscopy

We evaluated CNA35 as a collagen marker in healthy and atherosclerotic arteries of mice after both ex vivo and in vivo administration and as a molecular imaging agent for the detection of atherosclerosis. CNA35 conjugated with fluorescent Oregon Green 488 (CNA35/OG488) was administered ex vivo to mounted viable muscular (uterine), elastic (carotid), and atherosclerotic (carotid) arteries and fresh arterial rings. Two-photon microscopy was used for imaging. CNA35/OG488 labeling in healthy elastic arteries was compared with collagen type I, III, and IV antibody labeling in histologic sections. For in vivo labeling experiments, CNA35/OG488 was injected intravenously in C57BL6/J and apolipoprotein E(-/-) mice. Ex vivo CNA35/OG488 strongly labeled collagen in the tunica adventitia, media, and intima of muscular arteries. In healthy elastic arteries, tunica adventitia was strongly labeled, but labeling in tunica media and intima was prevented by endothelium and elastic laminae. Histology confirmed the affinity of CNA35 for type I, III, and IV collagen in arteries. Strong CNA35/OG488 labeling was found in atherosclerotic plaques. In vivo applied CNA35/OG488 minimally labeled the tunica intima of healthy carotid arteries. Atherosclerotic plaques in apolipoprotein E(-/-) mice exhibited large uptake. CNA35/OG488 imaging in organs revealed endothelium as a limiting barrier for in vivo uptake. CNA35/OG488 is a good molecular imaging agent for atherosclerosis.     

Measurements of pH, sucrose and potassium ions in the phloem sap of castor bean (Ricinus communis) plants

The composition of phloem sap, sampled at different heights along, the stem of castor bean ( Ricinus communis L. cv. Gibsonii) plants, was determined. A gradient in pH was observed; the highest pH values occurred near the shoot apex, decreasing towards the base of the stem. The sucrose content of the exudate exhibited a similar gradient. The concentration of potassium ions was highest near the uppermost, full-grown leaves, decreasing towards the apex and the base of the stem. The importance of these findings for the understanding of phloem translocation and unloading is discussed.     

Feasibility and relevance of compound strain imaging in non-stenotic arteries: comparison between individuals with cardiovascular diseases and healthy controls

Background

Compound strain imaging is a novel method to noninvasively evaluate arterial wall deformation which has recently shown to enable differentiation between fibrous and (fibro-)atheromatous plaques in patients with severe stenosis. We tested the hypothesis that compound strain imaging is feasible in non-stenotic arteries and provides incremental discriminative power to traditional measures of vascular health (i.e., distensibility coefficient (DC), central pulse wave velocity [cPWV], and intima-media thickness [IMT]) for differentiating between participants with and without a history of cardiovascular diseases (CVD).

Methods

Seventy two participants (60 ± 7 years) with non-stenotic arteries (IMT < 1.1 mm) were categorized in healthy participants (CON, n = 36) and CVD patients (n = 36) based on CVD history. Participants underwent standardised ultrasound-based assessment (DC, cPWV, and IMT) and compound strain imaging (radial [RS] and circumferential [CS] strain) in left common carotid artery. Area under receiver operating characteristics (AROC)-curve was used to determine the discriminatory power between CVD and CON of the various measures.

Results

CON had a significantly (P < 0.05) smaller carotid IMT (0.68 [0.58 to 0.76] mm) than CVD patients (0.76 [0.68 to 0.80] mm). DC, cPWV, RS, and CS did not significantly differ between groups (P > 0.05). A higher CS or RS was associated with a higher DC (CS: r = ?0.32;p < 0.05 and RS: r = 0.24;p < 0.05) and lower cPWV (CS: r = 0.24;p < 0.05 and RS: r = ?0.25;p < 0.05). IMT could identify CVD (AROC: 0.66, 95%-CI: 0.53 to 0.79), whilst the other measurements, alone or in combination, did not significantly increase the discriminatory power compared to IMT.

Conclusions

In non-stenotic arteries, compound strain imaging is feasible, but does not seem to provide incremental discriminative power to traditional measures of vascular health for differentiation between individuals with and without a history of CVD.

     

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C.     

Serum cartilage oligomeric matrix protein (COMP) decreases in rheumatoid arthritis patients treated with infliximab or etanercept

Changes in serum cartilage oligomeric matrix protein (COMP) were studied during a 6-month period from initiation of treatment of rheumatoid arthritis patients with either infliximab or etanercept, to elucidate whether the favourable results of tissue protection reported in clinical trials are corroborated by changing levels of circulating COMP. Rheumatoid arthritis patients commencing treatment with infliximab (N = 32) or etanercept (N = 17) were monitored in accordance with a structured protocol. Only patients who were not receiving glucocorticoids or who were on a stable dose of oral prednisolone (<10 mg daily) were included. Serum COMP was measured by a sandwich immunoassay based on two monoclonal antibodies against human COMP in samples obtained at treatment initiation and at 3 and 6 months. Serum COMP decreased at 3 months in both infliximab- and etanercept-treated patients (P < 0.001 and <0.005, respectively) and remained low at 6 months. There was no significant correlation between changes in or concentrations of serum COMP and serum C-reactive protein at any time point. A decrease in serum COMP was seen both in ACR20 responders (patients meeting the American College of Rheumatology criteria for 20% improvement) and in nonresponders. The pattern of changes of serum COMP, a marker for cartilage turnover, in these patient groups supports the interpretation that infliximab and etanercept have a joint protective effect. Serum COMP has potential as a useful marker for evaluating tissue effects of novel treatment modalities in rheumatoid arthritis.     

Molecular systematic analysis reveals cryptic tertiary diversification of a widespread tropical rain forest tree

The broad geographic range of many Neotropical rain forest tree species implies excellent dispersal abilities or range establishment that preceded the formation of current dispersal barriers. In order to initiate historical analyses of such widespread Neotropical trees, we sequenced the nuclear ribosomal spacer (ITS) region of Symphonia globulifera L. f. (Clusiaceae) from populations spanning the Neotropics and western Africa. This rain forest tree has left unmistakable Miocene fossils in Mesoamerica (15.5-18.2 Ma) and in South America ( approximately 15 Ma). Although marine dispersal of S. globulifera is considered improbable, our study establishes three marine dispersal events leading to the colonization of Mesoamerica, the Amazon basin, and the West Indies, thus supporting the paleontological data. Our phylogeographic analysis revealed the spatial extent of the three Neotropical S. globulifera clades, which represent trans-Andes (Mesoamerica+west Ecuador), cis-Andes (Amazonia+Guiana), and the West Indies. Strong phylogeographic structure found among trans-Andean populations of S. globulifera stands in contrast to an absence of ITS nucleotide variation across the Amazon basin and indicates profound regional differences in the demographic history of this rain forest tree. Drawing from these results, we provide a historical biogeographic hypothesis to account for differences in the patterns of beta diversity within Mesoamerican and Amazonian forests.     

Transformation in Escherichia coli: stages in the process.

Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.     

Trans-Golgi network and subapical compartment of HepG2 cells display different properties in sorting and exiting of sphingolipids

In HepG2 cells, the subapical compartment (SAC) is involved in the biogenesis of membrane polarity. By contrast, direct apical transport originating from the trans-Golgi network (TGN), which may contribute to polarity establishment, has been poorly defined in these cells. Thus, although newly synthesized sphingolipids can be directly transported from the TGN to the apical membrane, numerous apical resident proteins are traveling via the transcytotic route. Here, we developed an in vitro transport assay and compared the molecular sorting of 6-[N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)amino] hexanoyl-sphingomyelin (C(6)NBD-SM) and C(6)NBD-glucosylceramide (C(6)NBD-GlcCer) in TGN and SAC. SM is released from both TGN and SAC in the lumenal leaflet of transport vesicles. This holds also for GlcCer released from the SAC but not for a substantial fraction that departed from the Golgi. Distinct transport vesicles, enriched in either SM or GlcCer are released from SAC, consistent with their rigid sorting in this compartment. Different vesicle populations could not be recovered from TGN, although in situ experiments reveal that GlcCer is preferentially transported to the apical membrane, reflecting different transport mechanisms. The results indicate that in HepG2 cells sphingolipids are mainly sorted in the SAC membrane and that the release of SM from SAC and TGN is differentially regulated.     

Field-selected cadmium tolerance in the springtail Orchesella cincta is correlated with increased metallothionein mRNA expression

Populations of the springtail Orchesella cincta that live in metal contaminated soils have developed tolerance to cadmium by increased metal retention in the midgut epithelium and excretion at every moult. Regulation of the MT gene was studied in a tolerant population (Plombières, Belgium) and a laboratory culture. Animals were exposed to a range of concentrations of cadmium in the food (0-1.5 microM Cd/g food). RNA was extracted after 5-14 days of cadmium exposure and used for Northern blot analysis to quantify MT mRNA. MT expression levels were significantly higher (p <0.01) in individuals from laboratory-raised strains originating from the soils of the metal contaminated forest Plombières (2.4- to 7.8-fold expression) compared to the reference population (1.5- to 2.4-fold expression). No variable sites were found in the complete MT coding sequence. Southern blot analysis suggests that in both populations the gene is not tandemly repeated. This is the first evidence of evolution of metal tolerance via gene regulation of MT in a natural population. These data indicate a higher fitness of the tolerant population in the polluted environment due to selection of high MT expression phenotypes.