Gene expression of LOX-1, VCAM-1, and ICAM-1 in pre-atherosclerotic mice

To identify markers of the earliest stage of atherosclerosis, endothelial dysfunction, we evaluated the gene expression of lectin-like oxidized-low-density-lipoprotein receptor-1 (LOX-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in very young pre-atherosclerotic mice. Furthermore, the plasma levels of the soluble VCAM-1 and ICAM-1 were compared to the gene expression profiles. Gene expressions of LOX-1 and VCAM-1 were up-regulated in young apoE^−/− mice, and thus, it seems probable that these genes play a role in pre-atherosclerosis. Contrarily, the gene expression profile of ICAM-1 did not show any apparent differences between the groups, questioning the involvement of this molecule in the early development of atherosclerosis. Plasma levels of sVCAM-1 and sICAM-1 were similar in all mice and did not correlate with the vascular gene expression of the corresponding genes. It therefore seems likely that these circulating markers are not suited to detect early atherosclerosis.     

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.     

128-Slice Acceletated-Pitch Dual Energy CT Angiography of the Head and Neck: Comparison of Different Low Contrast Medium Volumes

Background

Our study aims to evaluate the image quality and feasibility of 128-slice dual-energy CTA (DE-CTA) for supra-aortic arteries using reduced amounts of contrast medium (CM).

Methods

A prospective study was performed in 54 patients receiving CTA of the head and neck with a 128-slice dual-source CT system. Patients were randomized into two groups with a volume of either 40 mL of CM (Group I) or 50 mL of CM (Group II). Arterial and venous enhancements were recorded for quantitative assessment. Qualitative assessments for images without bone removal (BR) were based on a) the visualization of the circle of Willis and b) streak artifacts due to residual CM in the subclavian or internal jugular veins ipsilateral to injection of CM. Qualitative assessment of dual-energy images using BR was based on the presence of bone remnants and vessel integrity. Quantitative data was compared using the Student t test. The χ² test was used for the qualitative measurements of streak artifacts in veins while the Mann-Whitney U test was used for the qualitative measurements of images with BR.

Results

Arterial and venous attenuation was significantly higher in Group II (P=0.000). Image quality regarding the circle of Willis was excellent in both groups (3.90±0.30 for Group I and 4.00±0 for Group II) . Imaging of the internal jugular veins was scored higher in Group I (1.87±0.72) compared with Group II (1.48±0.51) (P=0.021). Within Group I using BR, mean scores for bone remnants did not differ significantly (P>0.05) but mean scores of vessel integrity (P<0.05) did.

Conclusions

Contrast-enhanced head and neck CTA is feasible using a scan protocol with low amounts of contrast medium (40 mL) on a 128-slice dual-energy CTA. The 40-mL protocol provides satisfactory image quality before and after dual-energy bone-removal post-processing.     

CYP719B1 Is Salutaridine Synthase, the C-C Phenol-coupling Enzyme of Morphine Biosynthesis in Opium Poppy

Morphine is a powerful analgesic natural product produced by the opium poppy Papaver somniferum. Although formal syntheses of this alkaloid have been reported, the morphine molecule contains five stereocenters and a C-C phenol linkage that to date render a total synthesis of morphine commercially unfeasible. The C-C phenol-coupling reaction along the biosynthetic pathway to morphine in opium poppy is catalyzed by the cytochrome P450-dependent oxygenase salutaridine synthase. We report herein on the identification of salutaridine synthase as a member of the CYP719 family of cytochromes P450 during a screen of recombinant cytochromes P450 of opium poppy functionally expressed in Spodoptera frugiperda Sf9 cells. Recombinant CYP719B1 is a highly stereo- and regioselective enzyme; of forty-one compounds tested as potential substrates, only (R)-reticuline and (R)-norreticuline resulted in formation of a product (salutaridine and norsalutaridine, respectively). To date, CYP719s have been characterized catalyzing only the formation of a methylenedioxy bridge in berberine biosynthesis (canadine synthase, CYP719A1) and in benzo[c]phenanthridine biosynthesis (stylopine synthase, CYP719A14). Previously identified phenol-coupling enzymes of plant alkaloid biosynthesis belong only to the CYP80 family of cytochromes. CYP719B1 therefore is the prototype for a new family of plant cytochromes P450 that catalyze formation of a phenol-couple.The C-O or C-C phenol-couple is widely present in the plant kingdom in natural product biosynthetic processes such as alkaloid (1), lignan (2), lignin (3), and gallotannin (4) formation. Phenol-coupling reactions in nature were thought to be catalyzed by a variety of oxidative enzymes with broad substrate specificity such as peroxidases, polyphenol oxidases, and laccases. More recently, several enzymes discovered to be responsible for the formation of intermolecular C-O phenol and intramolecular C-C phenol-couples were found to be highly regio- and/or stereoselective catalysts. The first intermolecular C-O phenol-coupling enzyme identified was the cytochrome P450-dependent oxidase berbamunine synthase (CYP80A1) of bisbenzylisoquinoline alkaloid biosynthesis in Berberis cell cultures (5, 6) (Fig. 1). This enzyme is regiospecific, but will accept either (R)- and (S)-N-methylcoclaurine to form R-R and R-S phenol-coupled products. Absolute regio- and stereospecificity is demonstrated in the formation of the lignan (+)-pinoresinol from two molecules of coniferyl alcohol, a reaction guided by dirigent proteins that can be catalyzed by a range of oxidases or oxidants (7). The aporphine alkaloid intramolecular C-C phenol-couple is catalyzed in Coptis japonica cell cultures by the cytochrome P450-dependent oxidase CYP80G2; this enzyme accepts six tetrahydrobenzylisoquinoline alkaloids as substrate (8).Open in a separate windowFIGURE 1.Selected phenol-coupling reactions of alkaloid biosynthesis. Berbamunine synthase (CYP80A1) catalyzes the C-O intermolecular phenol-coupling reaction of bisbenzyisoquinoline alkaloid biosynthesis. (S)-Corytuberine synthase (CYP80G2) catalyzes formation of the intramolecular C-C phenol-couple in magnoflorine biosynthesis. Salutaridine synthase forms the C-C intramolecular phenol-couple of salutaridine in morphine biosynthesis.Morphine has often been described as the king of alkaloids. Although formal syntheses of this powerful analgesic have been reported, yields are low (Ref. 9 and references therein); attempts in organic chemistry to mimic the biosynthetic formation of the C-C phenol-couple of salutaridine (Fig. 1) have been either unsuccessful, yielding rather isoboldine or pallidine (10), or have resulted in very low yield of salutaridine (11) or in a mixture of isoboldine and salutaridine, with the reaction favoring formation of isoboldine by a factor of ∼5 (12). Along with the five stereocenters present in this molecule, the C-C phenol-couple renders a chemical synthesis of morphine commercially unfeasible. The enzyme catalyzing this reaction in planta was sought unsuccessfully for many years and was discovered finally in the opium poppy Papaver somniferum to be a cytochrome P450-dependent oxidase that stereo- and regiospecifically produces salutaridine by C-C phenol-coupling of (R)-reticuline (Fig. 1) (1, 13). The native enzyme salutaridine synthase was unstable, which precluded protein purification for further characterization.Herein, we describe the identification and functional expression of opium poppy salutaridine synthase, a member of the cytochrome P450 family, in Spodoptera frugiperda Sf9 cells. The recombinant enzyme was sufficiently stable in insect cell culture to be characterized with respect to substrate specificity and steady state kinetic values. Recombinant salutaridine synthase converted (R)-reticuline exclusively to salutaridine and (R)-norreticuline exclusively to norsalutaridine (N-demethylsalutaridine).     

Targeted inactivation of the murine Abca3 gene leads to respiratory failure in newborns with defective lamellar bodies

Mutations in the human ABCA3 gene, encoding an ABC-transporter, are associated with respiratory failure in newborns and pediatric interstitial lung disease. In order to study disease mechanisms, a transgenic mouse model with a disrupted Abca3 gene was generated by targeting embryonic stem cells. While heterozygous animals developed normally and were fertile, individuals homozygous for the altered allele (Abca3-/-) died within one hour after birth from respiratory failure, ABCA3 protein being undetectable. Abca3-/- newborns showed atelectasis of the lung in comparison to a normal gas content in unaffected or heterozygous littermates. Electron microscopy demonstrated the absence of normal lamellar bodies in type II pneumocytes. Instead, condensed structures with apparent absence of lipid content were found. We conclude that ABCA3 is required for the formation of lamellar bodies and lung surfactant function. The phenotype of respiratory failure immediately after birth corresponds to the clinical course of severe ABCA3 mutations in human newborns.     

Nitrogen yield advantage from grass–legume mixtures is robust over a wide range of legume proportions and environmental conditions

Current challenges to global food security require sustainable intensification of agriculture through initiatives that include more efficient use of nitrogen (N), increased protein self‐sufficiency through homegrown crops, and reduced N losses to the environment. Such challenges were addressed in a continental‐scale field experiment conducted over 3 years, in which the amount of total nitrogen yield (N_tot) and the gain of N yield in mixtures as compared to grass monocultures (N_gainmix) was quantified from four‐species grass–legume stands with greatly varying legume proportions. Stands consisted of monocultures and mixtures of two N₂‐fixing legumes and two nonfixing grasses. The amount of N_tot of mixtures was significantly greater (P ≤ 0.05) than that of grass monocultures at the majority of evaluated sites in all 3 years. N_tot and thus N_gainmix increased with increasing legume proportion up to one‐third of legumes. With higher legume percentages, N_tot and N_gainmix did not continue to increase. Thus, across sites and years, mixtures with one‐third proportion of legumes attained ~95% of the maximum N_tot acquired by any stand and had 57% higher N_tot than grass monocultures. Realized legume proportion in stands and the relative N gain in mixture (N_gainmix/N_tot in mixture) were most severely impaired by minimum site temperature (R = 0.70, P = 0.003 for legume proportion; R = 0.64, P = 0.010 for N_gainmix/N_tot in mixture). Nevertheless, the relative N gain in mixture was not correlated to site productivity (P = 0.500), suggesting that, within climatic restrictions, balanced grass–legume mixtures can benefit from comparable relative gains in N yield across largely differing productivity levels. We conclude that the use of grass–legume mixtures can substantially contribute to resource‐efficient agricultural grassland systems over a wide range of productivity levels, implying important savings in N fertilizers and thus greenhouse gas emissions and a considerable potential for climate change mitigation.     

Screening HIV‐1 antigenic peptides as receptors for antibodies and CD4 in allosteric nanosensors

We have analyzed the suitability of six antigenic peptides from several HIV‐1 structural proteins (namely gp41, gp120, p17, and p24), as anti‐HIV‐1 antibody receptors in an allosteric enzymatic biosensor. These peptides were inserted in a solvent‐exposed surface of Escherichia coli (E. coli) beta‐galactosidase by means of conventional recombinant DNA technology. The resulting enzymes were tested to allosterically respond to sera from HIV‐1‐infected individuals. Only stretches from gp41 and gp120 envelope proteins were able to transduce the molecular contact signal in the presence of immunoreactive sera. Intriguingly, the enzyme displaying the CD4 binding site segment KQFINMWQEVGKAMYAPP was activated by soluble CD4, suggesting that it produces conformational modifications on the allosteric enzyme as those occurring during antibody‐promoted induced fit. This fact is discussed in the context of the design of smart protein drugs and markers targeted to CD4⁺ cells. Copyright © 2009 John Wiley & Sons, Ltd.     

A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase—extensive moonlighting in mitochondrial tRNA biogenesis

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m¹G9 and m¹A9 formation. The ability of the mitochondrial tRNA:m¹R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing.     

Laboratory Studies of Parasite Transmission Aspects between Hydrobia spp. (Gastropoda) and Corophium volutator (Amphipoda)

Aspects of parasite transmission between Hydrobia spp. and Corophium volutator, first and second intermediate host of digenetic trematodes, were investigated under laboratory conditions. H. ventrosa is used as an intermediate host by several trematode species. Under laboratory conditions the most frequently observed emergence from H. ventrosa was of cercariae of Maritrema subdolum. The number of cercariae shed per day varied considerably. It was observed that 30 cercariae on average and up to 450 cercariae at maximum can emerge from a single H. ventrosa per day. Cercarial production continued until the death of the snails. The life-span of cercariae of the species M. subdolum decreased as the water temperature increased. It can be concluded that under natural conditions the cercariae, after emerging, have a maximum period of 1 day in which to seek out their second intermediate host C. volutator. Almost all specimens of C. volutator exposed to cercariae of the species M. subdolum died within the test period of 6 days. High average cercarial densities caused short life-spans (<50 h), while at lower densities longer survival times were possible. For cercariae of other Trematoda species, we were unable to find any equally clear evidence of a reduction in the survival rate of C. volutator within the test period. Mortality of C volutator, and other effects of infestation, as observed in our experiment, can be assumed to be a result of the penetration process of the cercariae but they also can be attributed to the mesocercariae.     

R-wave synchronised atrial pacing in pediatric patients with postoperative junctional ectopic tachycardia: the atrioventricular interval investigated by computational analysis and clinical evaluation

Background

R-wave synchronised atrial pacing is an effective temporary pacing therapy in infants with postoperative junctional ectopic tachycardia. In the technique currently used, adverse short or long intervals between atrial pacing and ventricular sensing (AP–VS) may be observed during routine clinical practice.

Objectives

The aim of the study was to analyse outcomes of R-wave synchronised atrial pacing and the relationship between maximum tracking rates and AP–VS intervals.

Methods

Calculated AP–VS intervals were compared with those predicted by experienced pediatric cardiologist.

Results

A maximum tracking rate (MTR) set 10 bpm higher than the heart rate (HR) may result in undesirable short AP–VS intervals (minimum 83 ms). A MTR set 20 bpm above the HR is the hemodynamically better choice (minimum 96 ms). Effects of either setting on the AP–VS interval could not be predicted by experienced observers. In our newly proposed technique the AP–VS interval approaches 95 ms for HR > 210 bpm and 130 ms for HR < 130 bpm. The progression is linear and decreases strictly (? 0.4 ms/bpm) between the two extreme levels.

Conclusions

Adjusting the AP–VS interval in the currently used technique is complex and may imply unfavorable pacemaker settings. A new pacemaker design is advisable to allow direct control of the AP–VS interval.