Improved Method for Detection of Methanotrophic Bacteria in Forest Soils by PCR

A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the α-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils. Received: 1 September 2000 / Accepted: 2 October 2000     

Selective inhibition of DNA gyrase in vitro by a GC specific eight-ring hairpin polyamide at nanomolar concentration

The influence of an eight-ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated. The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration. As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site. The pyrrole-imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti-gyrase active DNA binding agents.     

Antisense delivery using protamine-oligonucleotide particles

Protamine, a polycationic peptide (mol. wt 4000-4500), was evaluated as a potential penetration enhancer for phosphodiester antisense oligonucleotides (ODNs). Unique complexes in the form of nanoparticles were spontaneously formed, which we call 'proticles'. The stability of the particles and the ODNs bound into the proticles was examined in foetal calf serum and cell culture medium. FITC-labelled ODNs bound to protamine showed an increased cellular uptake into human histiocytic lymphoma U 937 cells compared to free ODNs. Proticles significantly decreased cellular growth in a cell proliferation assay using ODNs against the c- myc proto-oncogene.     

Molecular mechanics evaluation of the proposed mechanisms for the degradation of urea by urease

A thorough conformational search of all the conformations available to oxygen-bound urea within wild-type urease was carried out. Identical low energy urea conformations were obtained by a Ramachandran type plot for the NHis272-Ni1-O-Curea, and Ni1-O-Curea-Nurea dihedral angles. Ramachandran plots, with active sites and protonation states modified to model the different urease mechanisms, were used to evaluate the different mechanisms. Based upon the low energy conformations available to urea in the active site of wild-type urease one can conclude that the traditional "His320 acts as a base" mechanism is unlikely. while the N,O urea bridged and the reverse protonation mechanisms cannot be ruled out. A consensus hydrogen-bonding network that does not favor any of the mechanisms has been reconfirmed by the extensive conformational search.     

Computational analysis of the first biheterocyclization site of the antibiotic microcin B17

Microcin B 17 (MccB17) undergoes an enzyme catalyzed posttranslational modification to form four oxazole and four thiazole rings. Four of these rings form 4,2 - connected biheterocyclic functionalities. In this study, the hexapeptide sequence surrounding the first biheterocyclization site of microcin B17 was examined using computational calculations and database analysis to see if it was preorganized for cyclization in a manner similar to that found in the autocatalytic posttranslational cyclization of Green Fluorescent Protein (GFP). Attention was focused on the intermolecular distances between the sulfur and oxygen atoms of the cysteine and serine residues and the carbonyl carbons which they attack in the ring formation. Conformational searches located some low energy conformations that contained relatively short oxygen to carbonyl carbon distances, which indicated that the oxazole forming fragment in microcin B17 is preorganized for cyclization. However, the lack of any clear patterns for the sulfur to carbon distances show that the side-chain of cysteine does not adopt any low energy conformations that are geometrically preorganized for cyclization. The MccB17 synthetase enzyme complex which catalyzes the cyclization process therefore has both steric and electronic functions. The data obtained in this investigation is in agreement with empirical data which shows that biheterocyclization will only occur if the thiazole forms before the oxazole.     

Simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography–tandem mass spectrometry

Quantitative analysis of therapeutic compounds and their metabolites in biological matrix (such as plasma, serum or urine) nowadays requires sensitive and selective methods to allow the determination of concentrations in the ng/ml range. A new on-line LC–MS–MS method using atmospheric pressure chemical ionisation (APCI) as interface for the simultaneous determination of nifedipine (NIF) and its metabolite in human plasma, dehydronifedipine (DNIF) has been developed. The compounds were extracted from plasma using solid-phase extraction (SPE) on disposable extraction cartridges (DECs). The SPE operations were performed automatically by means of a sample processor equipped with a robotic arm (ASPEC system). The DEC filled with phenyl modified silica was first conditioned with methanol and water. The washing step was performed with water. Finally, the analytes were successively eluted with methanol and water. The liquid chromatographic (LC) separation of NIF and DNIF was achieved on a RP-18 stationary phase (4 μm). The mobile phase consisted of methanol–50 mM ammonium acetate solution (50:50, v/v). The LC was then coupled to tandem mass spectrometry with an APCI interface in the positive ion mode.

The method developed was validated. The absolute recoveries evaluated over the whole concentration range were 95±2% and 95±4% for NIF and DNIF, respectively. The method was found to be linear in the 0.5–100 ng/ml concentration range for the two analytes (r²=0.999 for both NIF and DNIF). The mean R.S.D. values for repeatability and intermediate precision were 2.9 and 3.0% for NIF and 2.2–4.7% for the metabolite.The method developed was successfully used to investigate the plasma concentration of NIF and DNIF in the pharmacokinetic studies.     

Permeation of Therapeutic Drugs in Different Formulations across the Airway Epithelium In Vitro

Background

Pulmonary drug delivery is characterized by short onset times of the effects and an increased therapeutic ratio compared to oral drug delivery. This delivery route can be used for local as well as for systemic absorption applying drugs as single substance or as a fixed dose combination. Drugs can be delivered as nebulized aerosols or as dry powders. A screening system able to mimic delivery by the different devices might help to assess the drug effect in the different formulations and to identify potential interference between drugs in fixed dose combinations. The present study evaluates manual devices used in animal studies for their suitability for cellular studies.

Methods

Calu-3 cells were cultured submersed and in air-liquid interface culture and characterized regarding mucus production and transepithelial electrical resistance. The influence of pore size and material of the transwell membranes and of the duration of air-liquid interface culture was assessed. Compounds were applied in solution and as aerosols generated by MicroSprayer IA-1C Aerosolizer or by DP-4 Dry Powder Insufflator using fluorescein and rhodamine 123 as model compounds. Budesonide and formoterol, singly and in combination, served as examples for drugs relevant in pulmonary delivery.

Results and Conclusions

Membrane material and duration of air-liquid interface culture had no marked effect on mucus production and tightness of the cell monolayer. Co-application of budesonide and formoterol, applied in solution or as aerosol, increased permeation of formoterol across cells in air-liquid interface culture. Problems with the DP-4 Dry Powder Insufflator included compound-specific delivery rates and influence on the tightness of the cell monolayer. These problems were not encountered with the MicroSprayer IA-1C Aerosolizer. The combination of Calu-3 cells and manual aerosol generation devices appears suitable to identify interactions of drugs in fixed drug combination products on permeation.     

Criteria Used in Clinical Practice to Guide Immunosuppressive Treatment in Patients with Primary Sclerosing Cholangitis

Background & Aims

Current guidelines recommend immunosuppressive treatment (IT) in patients with primary sclerosing cholangitis (PSC) and elevated aminotransferase levels more than five times the upper limit of normal and elevated serum IgG-levels above twice the upper limit of normal. Since there is no evidence to support this recommendation, we aimed to assess the criteria that guided clinicians in clinical practice to initiate IT in patients with previously diagnosed PSC.

Methods

This is a retrospective analysis of 196 PSC patients from seven German hepatology centers, of whom 36 patients had received IT solely for their liver disease during the course of PSC. Analyses were carried out using methods for competing risks.

Results

A simplified autoimmune hepatitis (AIH) score >5 (HR of 36, p<0.0001) and a modified histological activity index (mHAI) greater than 3/18 points (HR 3.6, p = 0.0274) were associated with the initiation of IT during the course of PSC. Of note, PSC patients who subsequently received IT differed already at the time of PSC diagnosis from those patients, who did not receive IT during follow-up: they presented with increased levels of IgG (p = 0.004) and more frequently had clinical signs of cirrhosis (p = 0.0002).

Conclusions

This is the first study which investigates the parameters associated with IT in patients with PSC in clinical practice. A simplified AIH score >5 and a mHAI score >3, suggesting concomitant features of AIH, influenced the decision to introduce IT during the course of PSC. In German clinical practice, the cutoffs used to guide IT may be lower than recommended by current guidelines.     

Physiological and molecular analysis of the interactive effects of feeding and high environmental ammonia on branchial ammonia excretion and Na+ uptake in freshwater rainbow trout

Recently, a “Na⁺/NH₄ ⁺ exchange complex” model has been proposed for ammonia excretion in freshwater fish. The model suggests that ammonia transport occurs via Rhesus (Rh) glycoproteins and is facilitated by gill boundary layer acidification attributable to the hydration of CO₂ and H⁺ efflux by Na⁺/H⁺ exchanger (NHE-2) and H⁺-ATPase. The latter two mechanisms of boundary layer acidification would occur in conjunction with Na⁺ influx (through a Na⁺ channel energized by H⁺-ATPase and directly via NHE-2). Here, we show that natural ammonia loading via feeding increases branchial mRNA expression of Rh genes, NHE-2, and H⁺-ATPase, as well as H⁺-ATPase activity in juvenile trout, similar to previous findings with ammonium salt infusions and high environmental ammonia (HEA) exposure. The associated increase in ammonia excretion occurs in conjunction with a fourfold increase in Na⁺ influx after a meal. When exposed to HEA (1.5 mmol/l NH₄HCO₃ at pH 8.0), both unfed and fed trout showed differential increases in mRNA expression of Rhcg2, NHE-2, and H⁺-ATPase, but H⁺-ATPase activity remained at control levels. Unfed fish exposed to HEA displayed a characteristic reversal of ammonia excretion, initially uptaking ammonia, whereas fed fish (4 h after the meal) did not show this reversal, being able to immediately excrete ammonia against the gradient imposed by HEA. Exposure to HEA also led to a depression of Na⁺ influx, demonstrating that ammonia excretion can be uncoupled from Na⁺ influx. We suggest that the efflux of H⁺, rather than Na⁺ influx itself, is critical to the facilitation of ammonia excretion.     

Angiotensin II triggers release of leukotriene C4 in vascular smooth muscle cells via the multidrug resistance-related protein 1

The multidrug resistance-related protein-1 (MRP1) is important for the management of oxidative stress in vascular cells in vivo. Substrates of MRP1 are, among others, glutathione and the leukotriene C₄ (LTC₄), an eicosanoid and mediator of inflammation. Angiotensin (Ang) II infusion results in MRP1^?/? mice compared to wild-type mice in improved endothelial function and reduced reactive oxygen species (ROS) formation. However, the interaction between Ang II, LTC₄ and MRP1 is not completely understood and has never been investigated in vitro. Ang II induced in vascular smooth muscle cells (VSMC) the release of LTC₄ and the generation of ROS. Pharmacologic inhibition of MRP1 via MK 571 significantly reduced Ang II-induced ROS release (L012-luminescence) in VSMC. The release of ROS after Ang II stimulation is inhibited, to a comparable degree, by blockade of the Cys-LT1 receptor with montelukast. Incubation of VSMC with recombined LTC₄ and Ang II caused enhanced rates of proliferation in VSMC. This effect can be rescued by either MRP1 or Cys-LT1 receptor inhibition. Accordingly, stimulation of VSMC with LTC₄ reduces intracellular levels of glutathione, but does not affect apoptosis. LTC₄ stimulation results in a significant activation of MRP1, but does not alter MRP1 expression. These findings indicate a connection between Ang II, MRP1 and LTC₄. Both, MRP1 and LTC₄, are potentially promising targets for atheroprotective therapy.