A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.     

The hydrolysis of the anti-cancer ruthenium complex NAMI-A affects its DNA binding and antimetastatic activity: an NMR evaluation

The coordination of the antimetastatic agent NAMI-A, [H(2)im][trans-RuCl(4)(dmso-S)(Him)], (Him=imidazole; dmso=dimethyl sulfoxide), to the DNA model base 9-methyladenine (9-MeAde) was investigated in water. NMR spectroscopy was first applied for the study of the molecular stability and hydrolysis of NAMI-A in aqueous solution over a range of pH (3.0-7.4) and chloride ion concentrations (0-1 M) at 37.0 degrees C. In physiological conditions (phosphate buffer, pH 7.4) NAMI-A disappears from the solution in 15 min due to chloride and dmso hydrolysis, leading to uncharacterised poly-oxo Ru species. Conversely, at lower pH (3.0-6.0) and in water (pH approximately 5.5), only a partial dmso hydrolysis occurs, slowly forming the [trans-RuCl(4)(H(2)O)(Him)](-) complex. This latter species coordinates to 9-MeAde (via the N7 of 9-MeAde), forming the [trans-RuCl(4)(9-MeAde)(Him)](-) complex. NAMI-A and [trans-RuCl(4)(H(2)O)(Him)](-) give comparable intracellular ruthenium concentrations and accumulate in KB cells (human mouth carcinoma) and accumulate these at the G(2)/M phase, while poly-oxo Ru species do not, and their cell uptake is reduced to 50%. On the contrary, G(2)/M arrest and protein content in the murine metastatic cell line metGM, are not influenced by NAMI-A hydrolysis. Hydrolysed NAMI-A species apparently are easier taken up by the metGM cells, showing intracellular ruthenium concentrations one order of magnitude greater than those of intact NAMI-A. Therefore, it is proposed that the selective antimetastatic activity of NAMI-A during in vivo experiments can be attributed to its hydrolysed species.     

A high-resolution map of the H1 locus harbouring resistance to the potato cyst nematode Globodera rostochiensis

The resistance gene H1 confers resistance to the potato cyst nematode Globodera rostochiensis and is located at the distal end of the long arm of chromosome V of potato. For marker enrichment of the H1 locus, a bulked segregant analysis (BSA) was carried out using 704 AFLP primer combinations. A second source of markers tightly linked to H1 is the ultra-high-density (UHD) genetic map of the potato cross SH × RH. This map has been produced with 387 AFLP primer combinations and consists of 10,365 AFLP markers in 1,118 bins (). Comparing these two methods revealed that BSA resulted in one marker/cM and the UHD map in four markers/cM in the H1 interval. Subsequently, a high-resolution genetic map of the H1 locus has been developed using a segregating F₁ SH × RH population consisting of 1,209 genotypes. Two PCR-based markers were designed at either side of the H1 gene to screen the 1,209 genotypes for recombination events. In the high-resolution genetic map, two of the four co-segregating AFLP markers could be separated from the H1 gene. Marker EM1 is located at a distance of 0.2 cM, and marker EM14 is located at a distance of 0.8 cM. The other two co-segregating markers CM1 (in coupling) and EM15 (in repulsion) could not be separated from the H1 gene.Communicated by J.G. Wenzel     

Determination of midazolam in human plasma by liquid chromatography with mass-spectrometric detection

A liquid chromatographic assay with mass-spectrometric detection was developed for the quantitative determination of the cytochrome p450 3A phenotyping probe midazolam in human plasma. Sample pretreatment involved a one-step extraction of 600 microl aliquots with ethyl acetate. Midazolam and the internal standard, lorazepam, were separated on a column (150 mm x 4.6mm, i.d.) packed with 5 microm Zorbax Eclipse XDB-C8 material, using a mobile phase composed of methanol and 10mM aqueous ammonium acetate (60:40, v/v). Column effluents were analyzed using mass-spectrometry with an atmospheric pressure chemical ionization source. Calibration curves were linear in the concentration range of 1.00-200 ng/ml. The accuracy and precision ranged from 92.8 to 112% and 0.056 to 13.4%, respectively, for four different concentrations of quality control samples analyzed in triplicate on eight separate occasions. The developed method was subsequently applied to study the pharmacokinetics of midazolam in a group of 35 human subjects at a single dose of 25 microg/kg.     

The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E

Anaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus, because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosome-bearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.     

Smooth muscle F-actin disassembly and RhoA/Rho-kinase signaling during endotoxin-induced alterations in pulmonary arterial compliance

Endotoxemia is associated with changed pulmonary vascular function with respect to vasoreactivity, endothelial permeability, and activation of inducible nitric oxide synthase II (NOSII). However, whether altered passive arterial wall mechanics contribute to this endotoxin-induced pulmonary vascular dysfunction is still unknown. Therefore, we investigated whether endotoxin affects the passive arterial mechanics and compliance of isolated rat pulmonary arteries. Pulmonary arteries of pentobarbital-anesthetized Wistar rats (n = 55) were isolated and exposed to Escherichia coli endotoxin (50 microg/ml) for 20 h. Endotoxin increased pulmonary artery diameter and compliance (transmural pressure = 13 mmHg) in an endothelium-, Ca2+-, or NOSII-induced NO release-independent manner. Interestingly, the endotoxin-induced alterations in the passive arterial mechanics were accompanied by disassembly of the smooth muscle cell (SMC) F-actin cytoskeleton. Disassembly of F-actin by incubation of control arteries with the cytoskeleton-disrupting agent cytochalasin B or the Rho-kinase inhibitor Y-27632 induced a similar increase in passive arterial diameter and compliance. In contrast, RhoA activation by lysophosphatidic acid prevented the endotoxin-induced alterations in the pulmonary SMC F-actin cytoskeleton and passive mechanics. In conclusion, these findings indicate that disassembly of the SMC F-actin cytoskeleton and RhoA/Rho-kinase signaling act as mediators of endotoxin-induced changes in the pulmonary arterial mechanics. They imply the involvement of F-actin rearrangement and RhoA/Rho-kinase signaling in endotoxemia-induced vascular lung injury.     

Systematic adjudication of myocardial infarction end-points in an international clinical trial

Background

Clinical events committees (CEC) are used routinely to adjudicate suspected end-points in cardiovascular trials, but little information has been published about the various processes used. We reviewed results of the CEC process used to identify and adjudicate suspected end-point (post-enrolment) myocardial infarction (MI) in the large Platelet Glycoprotein IIb/IIIa in Unstable Angina: Receptor Suppression Using Integrilin (Eptifibatide) Therapy (PURSUIT) trial.

Methods

The PURSUIT trial randomised 10,948 patients with acute coronary syndromes to receive eptifibatide or placebo. A central adjudication process was established prospectively to identify all suspected MIs and adjudicate events based on protocol definitions of MI. Suspected MIs were identified by systematic review of data collection forms, cardiac enzyme results, and electrocardiograms. Two physicians independently reviewed all suspected events. If they disagreed whether a MI had occurred, a committee of cardiologists adjudicated the case.

Results

The CEC identified 5005 patients with suspected infarction (46%), of which 1415 (28%) were adjudicated as end-point infarctions. As expected, the process identified more end-point events than did the site investigators. Absolute and relative treatment effects of eptifibatide were smaller when using CEC-determined MI rates rather than site investigator-determined rates. The site-investigator reporting of MI and the CEC assessment of MI disagreed in 20% of the cases reviewed by the CEC.

Conclusions

End-point adjudication by a CEC is important, to provide standardised, systematic, independent, and unbiased assessment of end-points, particularly in trials that span geographic regions and clinical practice settings. Understanding the CEC process used is important in the interpretation of trial results and event rates.     

Routine DNA analysis based on 12S rRNA gene sequencing as a tool in the management of captive primates

Automated DNA sequencing of a fragment of the relatively slowly evolving mitochondrial 12S rRNA gene was used to distinguish primate species, and the method was compared with species determination based upon classical taxonomy. DNA from blood from 53 monkeys housed at the Stichting AAP Shelter for Exotic Animals, all Old World monkeys, was amplified by polymerase chain reaction (PCR) with a primer set spanning approximately 390 nucleotides of the mitochondrial 12S rRNA gene. The products were directly sequenced and compared with our database of primate 12S sequences. Many individuals were found to harbor a 12S sequence identical to one of the reference sequences. For others, phylogenetic methods were used for species estimation, which was especially informative in Cercopithecus species.     

Clinical and angiographic results with the NIR stent: First International NIR Endovascular Stent Study (FINESS-II)

BACKGROUND: Although safety and efficacy of the NIR trade mark stent have been reported, the long-term angiographic and clinical outcomes have yet to be investigated. The FINESS-II study (First International NIR Endovascular Stent Study) was designed to assess the procedural safety of single 9 and 16 mm NIR stent implantation, the six-month restenosis rate and finally the six- and 12-month clinical outcome of patients treated with this novel coronary stent. METHODS: Patients with angina and a single de novo lesion in a native coronary artery of >3 and <5 mm diameter were included in this multicentre, prospective, observational trial. Clinical follow-up was obtained at one, six and 12 months. Angiography was performed before and after the stent implantation and at six months. The primary endpoint included major adverse cardiac events (death, myocardial infarction and target lesion revascularization) within 30 days after the procedure. Major bleeding complications and subacute stent thrombosis within the first 30 days were also reported as specific endpoints. Secondary endpoints were major cardiac-event-free survival at six- and 12-month follow-up and angiographic restenosis at six months. RESULTS: A total of 156 patients (81% male, mean age 60 +/- 10 years), with stable (54%), unstable (40%) angina pectoris or silent ischemia (6%) were enrolled. The target vessel diameter was 2.94 +/- 0.54 mm. The minimal lumen diameter pre, post and at follow-up was 1.04 +/- 0.32 mm, 2.64 +/- 0.42 mm and 1.88 +/- 0.63 mm, respectively. Restenosis rate according to the >50% diameter stenosis criterion at six month follow-up was 19% (26/136). At 12 months, the event-free survival rate was 83% (two deaths, one Q-wave and three non-Q-wave myocardial infarctions, four bypass surgery and 17 target lesion revascularizations), while 87% of the patients were free of angina pectoris. CONCLUSION: the outcome of the FINESS-II trial is comparable to those observed in previous stent trials (Benestent II), indicating that the coronary NIR stent is safe and effective as a primary device for the treatment of native coronary artery lesions in patients with (un)stable angina pectoris.