Likely Introduction Date of Schmallenberg Virus into France According to Monthly Serological Surveys in Cattle.

To estimate the date of introduction of Schmallenberg virus (SBV) into France, the prevalence of antibodies against the virus was determined monthly in cattle from two northern departments from August 2011 to April 2012. Seropositive cattle were detected from October 2011 in both departments with a prevalence of 55.6% in the westernmost department (Meurthe-et-Moselle) and of 12.7% in the easternmost department (Manche). Schmallenberg virus seroprevalence then increased rapidly to high levels.

The lipopeptide antibiotic Friulimicin B inhibits cell wall biosynthesis through complex formation with bactoprenol phosphate.

Friulimicin B is a naturally occurring cyclic lipopeptide, produced by the actinomycete Actinoplanes friuliensis, with excellent activity against gram-positive pathogens, including multidrug-resistant strains. It consists of a macrocyclic decapeptide core and a lipid tail, interlinked by an exocyclic amino acid. Friulimicin is water soluble and amphiphilic, with an overall negative charge. Amphiphilicity is enhanced in the presence of Ca(2+), which is also indispensable for antimicrobial activity. Friulimicin shares these physicochemical properties with daptomycin, which is suggested to kill gram-positive bacteria through the formation of pores in the cytoplasmic membrane. In spite of the fact that friulimicin shares features of structure and potency with daptomycin, we found that friulimicin has a unique mode of action and severely affects the cell envelope of gram-positive bacteria, acting via a defined target. We found friulimicin to interrupt the cell wall precursor cycle through the formation of a Ca(2+)-dependent complex with the bactoprenol phosphate carrier C(55)-P, which is not targeted by any other antibiotic in use. Since C(55)-P also serves as a carrier in teichoic acid biosynthesis and capsule formation, it is likely that friulimicin blocks multiple pathways that are essential for a functional gram-positive cell envelope.

Exploiting the genetic potential of polyketide producing streptomycetes.

Streptomycetes are the most important bacterial producers of bioactive secondary metabolites such as antibiotics or cytostatics. Due to the emerging resistance of pathogenic bacteria to all commonly used antibiotics, new and modified natural compounds are required for the development of novel drugs. In addition to the classical screening for natural compounds, genome driven approaches like combinatorial biosynthesis are permanently gaining relevance for the generation of new structures. This technology utilizes the combination of genes from different biosynthesis pathways resulting in the production of novel or modified metabolites. The basis for this strategy is the access to a significant number of genes and the knowledge about the activity and specificity of the enzymes encoded by them. A joint initiative was started to exploit the biosynthesis gene clusters from streptomycetes. In this publication, an overview of the strategy for the identification and characterization of numerous biosynthesis gene clusters for polyketides displaying interesting functions and particular structural features is given.

Biosynthetic gene cluster of simocyclinone, a natural multihybrid antibiotic.

The entire simocyclinone biosynthetic cluster (sim gene cluster) from the producer Streptomyces antibioticus Tü6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the sim gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the sim gene cluster had been cloned.

Development of three different gene cloning systems for genetic investigation of the new species Amycolatopsis japonicum MG417-CF17, the ethylenediaminedisuccinic acid producer.

For the first time gene cloning systems have been developed for Amycolatopsis japonicum. Direct transformation, polyethyleneglycol (PEG) induced protoplast transformation and conjugal transfer was established for A. japonicum MG417-CF17, the ethylenediaminedisuccinic acid (EDDS) producer. The direct transformation procedure was modified to introduce DNA. The most important parameter for an efficient DNA uptake was the age of the culture. Using of mycelium from 36-h old cultures resulted in the highest transformation frequencies. Further, conditions for transformation of A. japonicum protoplasts were established. The efficiency of transformation depended mainly on the source of PEG and the components of the regeneration agar. The replicative plasmid pULVK2A carrying pA-rep and the apramycin resistance gene was transferred into the EDDS producer with a frequency of 0.38 colonies microg(-1) DNA by using the direct transformation procedure and with a frequency of 0.56 colonies microg(-1) DNA by using the PEG induced protoplast transformation. The plasmid was genetically stable, and could easily be reisolated from A. japonicum. We also demonstrated that conjugal transfer of the plasmid pSET152 from Escherichia coli ET12567 (pUB307) to Amycolatopsis spores is possible. The plasmid pSET152 integrated in the A. japonicum chromosome. A titre of 2.4 x 10(-4) exconjugants per recipient was obtained.

Phosphorylation-dependent modulation of cardiac calcium current by intracellular free magnesium.

We compared the effects of cytosolic free magnesium (Mg(2+)(i)) on L-type Ca(2+) current (I(Ca,L)) in patch-clamped guinea pig ventricular cardiomyocytes under basal conditions, after inhibition of protein phosphorylation, and after stimulation of cAMP-mediated phosphorylation. Basal I(Ca,L) density displayed a bimodal dependence on the concentration of Mg(2+)(i) ([Mg(2+)](i); 10(-6)-10(-2) M), which changed significantly as cell dialysis progressed due to a pronounced and long-lasting rundown of I(Ca,L) in low-Mg(2+) dialysates. Ten minutes after patch breakthrough, I(Ca,L) density (at +10 mV) in Mg(2+)(i)-depleted cells ([Mg(2+)](i) approximately 1 microM) was elevated, increased to a maximum at approximately 20 microM [Mg(2+)](i), and declined steeply at higher [Mg(2+)](i). Treatment with the broad-spectrum protein kinase inhibitor K252a (10 microM) reduced I(Ca,L) density and abolished these effects of Mg(2+)(i) except for a negative shift of I(Ca,L)-voltage relations with increasing [Mg(2+)](i). Maximal stimulation of cAMP-mediated phosphorylation occluded the Mg(2+)(i)-induced stimulation of I(Ca,L) and prevented inhibitory effects of the ion at [Mg(2+)](i) <1 mM but not at higher concentrations. These results show that the modulation of I(Ca,L) by Mg(2+)(i) requires protein kinase activity and likely originates from interactions of the ion with proteins involved in the regulation of protein phosphorylation/dephosphorylation. Stimulatory effects of Mg(2+)(i) on I(Ca,L) seem to increase the cAMP-mediated phosphorylation of Ca(2+) channels, whereas inhibitory effects of Mg(2+)(i) appear to curtail and/or reverse cAMP-mediated phosphorylation.

Optical biosensors. Monitoring studies of glycopeptide antibiotic fermentation using white light interference.

This paper describes the design, characterization, and use of an optical biosensor suited for the process control of biotechnological processes. The detector principle is based on reflectometric interference spectroscopy (RIfS). RIfS enables a label-free, product-specific monitoring, with a future outline for on-line process control. The potential of the RIfS biosensor is exemplified by the qualitative and quantitative monitoring of the microbial production of vancomycin-type glycopeptide antibiotics.

A polyketide synthase in glycopeptide biosynthesis: the biosynthesis of the non-proteinogenic amino acid (S)-3,5-dihydroxyphenylglycine.

Balhimycin, a vancomycin-type antibiotic from Amycolatopsis mediterranei, contains the unusual amino acid (S)-3,5-dihydroxyphenylglycine (Dpg), with an acetate-derived carbon backbone. After sequence analysis of the biosynthetic gene cluster, one gene, dpgA, for a predicted polyketide synthase (PKS) was identified, sharing 20-30% identity with plant chalcone synthases. Inactivation of dpgA resulted in loss of balhimycin production, and restoration was achieved by supplementation with 3,5-dihydroxyphenylacetic acid, which is both a possible product of a PKS reaction and a likely precursor of Dpg. Enzyme assays with the protein expressed in Streptomyces lividans showed that this PKS uses only malonyl-CoA as substrate to synthesize 3,5-dihydroxyphenylacetic acid. The PKS gene is organized in an operon-like structure with three downstream genes that are similar to enoyl-CoA-hydratase genes and a dehydrogenase gene. The heterologous co-expression of all four genes led to accumulation of 3,5-dihydroxyphenylglyoxylic acid. Therefore, we now propose a reaction sequence. The final step in the pathway to Dpg is a transamination. A predicted transaminase gene was inactivated, resulting in abolished antibiotic production and accumulation of 3,5-dihydroxyphenylglyoxylic acid. Interestingly, restoration was only possible by simultaneous supplementation with (S)-3,5-dihydroxyphenylglycine and (S)-4-hydroxyphenylglycine, indicating that the transaminase is essential for the formation of both amino acids.

[Lipoprotein and apolipoprotein electrophoresis in X-chromosome recessive ichthyosis]. / Lipoprotein- und Apolipoproteinelektrophorese bei X-chromosomalrezessiver Ichthyose.

BACKGROUND AND OBJECTIVE: The clinical differentiation of the hereditary ichthyosis forms is difficult and without laboratory markers hardly possible. Serum lipoprotein electrophoresis is one tool for detecting patients with recessive X-linked ichthyosis (XRI). Compared to controls, XRI patients show elevated electrophoretic mobilities of low density (LDL) and very low density lipoproteins (VLDL). This change in pattern is only partially explained by the increased LDL cholesterin sulfate concentration and is the subject of this study. PATIENTS/METHODS: Patients suffering from XRI and ichthyosis vulgaris, healthy controls. SDS-PAGE-electrophoresis and isoelectric focusing for detection of XRI-associated variations in apolipoproteins apo B-100, apo C-III and apo E. RESULTS: XRI-associated apolipoprotein variants were not found. In contrast to the literature, an increased electrophoretic mobility was also observed for HDL (high density lipoproteins) from XRI patients. CONCLUSIONS: The underlying cause of the increased electrophoretic mobility of VLDL and HDL in XRI patients remains unclear. Future studies should investigate other apolipoproteins and verify the cholesterin sulfate concentrations reported for VLDL and HDL from XRI patients.

[Characterization of mechanical in vitro hemolysis and sub-hemolysis. 2: Variables of state and dimensionless characteristic values of hemolysis]. / Charakterisierung der mechanischen In-vitro-Hämolyse und -Subhämolyse. Teil 1: Zustandsgrössen und dimensionslose Kennzahlen der Hämolyse.

Blood damaging effects of artificial perfusion devices such as assist devices, heart valve prostheses, for example, must be evaluated in vitro before being used in the clinical setting. For this purpose, mainly animal blood has been used, and a number of associated problems are currently being discussed. Differences in the use of the term hemolysis--meaning breakdown of erythrocytes or increased plasma hemoglobin, result in incompatibility among different authors. In addition, subhemolytic damage and its quantification has not been investigated to any extent. Another problem are the differences in the mechanical fragility of erythrocytes from different animal species, and the question of transferability to the in vivo situation. Furthermore, the variability of mechanical stability within a given species is often greater than the differences between one species and another. International efforts are now being made to standardize haemolytic test conditions and the present study is meant as a contribution to this. In the first part we describe an extension of our LYSE number model. Characteristically, the model uses dimensionless similarity numbers, LY and MY, thus making the results obtained under different test conditions comparable with one another. The LY number reflects the breakdown of cells (decreasing hematocrit), the MY number an increase in plasma hemoglobin. Differences between LY and MY are an indication of subhemolytic events.

Semi-automated rapid isoelectric focusing of apolipoproteins C from human plasma using Phastsystem and immunofixation.

Apolipoproteins (apo) C-I, C-II, and C-III play crucial roles in intravascular lipid metabolism. Whereas apo C-II is an obligate cofactor for lipoprotein lipase, apo C-III was shown to inhibit its action. Apo C-I can be a potent cofactor of human lecithin:cholesterol acyltransferase. Structural mutants and deficiencies of apo C-II lead to hypertriglyceridemia. A similar phenotype is associated with apo C-III mutants and is inducible by overexpression of human apo C-III in transgenic animals. No structural variant has so far been reported for apo C-I. The present paper describes a rapid semi-automated procedure for isoelectric focusing analysis of these C-apolipoproteins from whole plasma or serum and their visualization by immunofixation and silver staining. The procedure allows detection of charged variants of C-apolipoproteins. As applied to 295 patients with coronary heart disease and 85 controls, it also serves to detect deficiency syndromes of these apolipoproteins. The procedure provides reliable, easy and quick analysis of C-apolipoproteins applicable as a routine or screening procedure not restricted to specialized laboratories.

Identification and analysis of the balhimycin biosynthetic gene cluster and its use for manipulating glycopeptide biosynthesis in Amycolatopsis mediterranei DSM5908.

Seven complete genes and one incomplete gene for the biosynthesis of the glycopeptide antibiotic balhimycin were isolated from the producer, Amycolatopsis mediterranei DSM5908, by a reverse-cloning approach and characterized. Using oligonucleotides derived from glycosyltransferase sequences, a 900-bp glycosyltransferase gene fragment was amplified and used to identify a DNA fragment of 9,882 bp. Of the identified open reading frames, three (oxyA to -C) showed significant sequence similarities to cytochrome P450 monooxygenases and one (bhaA) showed similarities to halogenase, and the genes bgtfA to -C showed similarities to glycosyltransferases. Glycopeptide biosynthetic mutants were created by gene inactivation experiments eliminating oxygenase and glycosyltransferase functions. Inactivation of the oxygenase gene(s) resulted in a balhimycin mutant (SP1-1) which was not able to synthesize an antibiotically active compound. Structural analysis by high-performance liquid chromatography-mass spectrometry, fragmentation studies, and amino acid analysis demonstrated that these oxygenases are involved in the coupling of the aromatic side chains of the unusual heptapeptide. Mutant strain HD1, created by inactivation of the glycosyltransferase gene bgtfB, produced at least four different compounds which were not glycosylated but still antibiotically active.

Isolation and characterization of the PEP-phosphomutase and the phosphonopyruvate decarboxylase genes from the phosphinothricin tripeptide producer Streptomyces viridochromogenes Tü494.

The previously isolated non-phosphinothricin tripeptide producing Streptomyces viridochromogenes gene disruption mutant SP62/2 was used to identify and analyze genes encoding early steps of the phosphinothricin tripeptide biosynthesis. Cross-feeding and bioconversion experiments between SP62/2 and known non-phosphinothricin tripeptide producing mutants or presumptive phosphinothricin tripeptide precursors revealed that SP62/2 was blocked in step one or two of the phosphinothricin tripeptide biosynthesis. It was shown that the block in the biosynthesis is due to the integration of a temperature-sensitive plasmid by illegitimate recombination into the phosphinothricin tripeptide biosynthetic gene cluster. The corresponding region was isolated from the wild-type. A 2.7-kb DNA fragment was analyzed comprising three ORFs (ppm, ppd, orfX) which are probably translationally coupled. The ppm gene encodes a protein which is similar to PEP-phosphomutases and the deduced Ppd product shows similarity to the phosphonopyruvate decarboxylase from Streptomyces wedmorensis.

[Consultation psychiatry in a university clinic--from the viewpoint of psychoanalytic supervision work]. / Konsiliarpsychiatrie in einer Universitätsklinik--aus dem Blickwinkel tiefenpsychologischer Supervisionsarbeit.

In contrast to English-speaking countries, Consultant/Liaison Psychiatry has been a neglected discipline in the GFR until now. Liaison Psychiatry in particular, is hardly represented. Consultant Psychiatry confronts the psychiatrist with a wide spectrum range of problems, whereas in his further training in the GFR he is insufficiently prepared for these responsibilities. Therefore, the authors report on a model which has already been tried and tested at the University Hospital in Ulm. Based on the annual data evaluation the authors outline the different questions in psychiatric consultations which are mainly raised and required by the Medical Hospital. In most cases they can be solved in case-focussed team conferences. However, within the framework of interdisciplinary cooperation there are more complex problems solved by regular psychotherapeutic supervision meetings with the whole team. The conditions for this procedure are clearly stated and illustrated by case studies.

Cloning and analysis of a peptide synthetase gene of the balhimycin producer Amycolatopsis mediterranei DSM5908 and development of a gene disruption/replacement system.

A gene cloning system for Amycolatopsis mediterranei DSM5908, the producer of the glycopeptide antibiotic balhimycin, was developed for analysis of peptide synthetase genes. A modified direct transformation procedure was used to introduce DNA. The efficiency of DNA uptake depended on the age of the culture: mycelium of early stationary phase (52-55 h) cultures resulted in optimal transformation frequencies. Using the novel non-replicative integration vector pSP1, gene disruption plasmids were constructed. Highest integration frequencies were observed when the DNA was isolated from the dam/dcm Escherichia coli strain JM110. The efficiency of integration depended directly on the size of the cloned insert. Plasmids with fragments smaller than 1 kilobase (kb) were difficult to integrate. In gene replacement experiments a high double cross-over rate (31%) was demonstrated. Oligonucleotides derived from conserved regions of peptide synthetases were designed to identify balhimycin biosynthesis genes. Using these gene probes in plaque hybridization experiments, we identified peptide synthetase homologous DNA fragments in a lambda library of A. mediterranei. One peptide synthetase gene fragment was characterized by DNA sequencing and the results revealed a complete amino acid activating domain of a peptide synthetase gene, designated aps. The disruption of aps neither influenced balhimycin biosynthesis nor generated another apparent phenotype.

Modulation of L-type Ca2+ current by fast and slow Ca2+ buffering in guinea pig ventricular cardiomyocytes.

Free Ca2+ near Ca2+ channel pores is expected to be lower in cardiomyocytes dialyzed with bis-(o-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) than with ethyleneglycol-bis-(beta-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) because BAPTA chelates incoming Ca2+ more rapidly. The consequences of intracellular Ca2+ buffering by BAPTA (0.2-60 mM) and by EGTA (0.2-67 mM) on whole-cell L-type Ca2+ current (ICa,L) were investigated in voltage-clamped guinea pig ventricular cardiomyocytes; bulk cytoplasmic free Ca2+ (Cac2+) was monitored using the fluorescent Ca2+ indicator indo-1. ICa,L was augmented by approximately 12-fold when BAPTA in the cell dialysate was increased from 0.2 to 50 mM (half-maximal stimulation at 31 mM), whereas elevating internal EGTA from 0.2 to 67 mM increased ICa,L only by approximately 2-fold. Cac2+ was < 20 nM with internal BAPTA or EGTA > or = 20 mM. While EGTA up to 67 mM had only an insignificant inhibitory effect on the stimulation of ICa,L by 3 microM forskolin, ICa,L in 50 mM BAPTA-dialyzed myocytes was insensitive to forskolin-induced elevation of adenosine 3',5'-cyclic monophosphate (cAMP); conversely, ICa,L in cAMP-loaded cells was unresponsive to BAPTA dialysis. Cell dialysis with BAPTA, but not with EGTA, accelerated the slow component of ICa,L inactivation (tau S) without affecting its fast component (tau F), resembling the effects of cAMP-dependent phosphorylation. BAPTA-stimulated ICa,L was inhibited by acetylcholine and by the cAMP-dependent protein kinase (PKA) blocker H-89. These results suggest that BAPTA-induced lowering of peri-channel Ca2+ stimulates cAMP synthesis and channel phosphorylation by disinhibiting Ca(2+)-sensitive adenylyl cyclase.

Beta-adrenoceptor-coupled Gs protein facilitates the activation of cAMP-dependent cardiac Cl- current.

Here a comparison is made between adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- current (ICl) density and activation time course in response to beta-adrenoceptor stimulation with isoproterenol and adenylyl cyclase activation with forskolin. Saturating concentrations of isoproterenol and forskolin failed to activate an ICl in guinea pig atrial as well as in rat and frog ventricular cardiomyocytes. In guinea pig ventricular cardiomyocytes, step application of 1 microM isoproterenol induced an ICl of -0.89 +/- 0.32 pA/pF (holding potential -40 mV, temperature 22 +/- 1 degrees C). ICl activation started after 3 +/- 1 s, was complete within 44 +/- 9 s, and was abolished after cell dialysis with the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate. Stimulation with increasing concentrations of forskolin (0.01-10 microM) increased ICl density and accelerated ICl activation. With 1 microM forskolin, ICl density was maximal (-0.57 +/- 0.30 pA/pF) but significantly smaller than that achieved with 1 microM isoproterenol. Although ICl density could not be further augmented by forskolin > 1 microM, current activation (latency 28 +/- 8 s, full activation after 112 +/- 8 s with 1 microM forskolin) was further accelerated by 3 and 10 microM forskolin. However, ICl activation with 10 microM forskolin was still slower than that with 1 microM isoproterenol. A low isoproterenol concentration (1 nM), which did not activate ICl by itself, accelerated the 1 microM forskolin-induced activation of ICl by 35%; this speeding up was abolished after cell dialysis with guanosine 5'-O-(2-thiodiphosphate). ICl deactivation after the washout of 1 microM forskolin or 1 microM isoproterenol followed a similar time course. After stimulation with 10 microM forskolin or 1 microM forskolin + 1 microM isoproterenol, but not with 1 microM forskolin + 1 nM isoproterenol, the decay of ICl was significantly delayed. These results indicate that both cAMP-dependent and cAMP-independent G protein pathways contribute to the regulation of guinea pig ventricular ICl.

Cyclic AMP-independent inhibition of cardiac calcium current by forskolin.

Low-to-moderate concentrations (< or = 3 microM) of forskolin (FSK) stimulated L-type Ca2+ current (ICa,L) and activated Cl- current (IC1) in guinea pig ventricular myocytes investigated under standard whole-cell conditions at 35 degrees. These stimulatory effects reached a steady state after several minutes and smoothly decayed after a short lag period on removal of the drug. Short (2-3 min) exposures to higher concentrations (10-100 microM) of FSK frequently had a multiphasic effect on ICa,L; marked stimulation during the first minute quickly faded during the next 1-2 min, and removal of the drug caused secondary stimulation that lasted for several minutes. Because the amplitude of cAMP-dependent ICl remained stable during the fade and secondary stimulation of ICa,L, the latter modulation of ICa,L seemed to be the result of a cAMP-independent inhibitory action of FSK on Ca2+ channels. Under conditions in which the stimulation of cAMP by FSK was slowed (22 degrees), rapid application of 10-30 microM FSK revealed that inhibition occurred within < 1 sec. In myocytes dialyzed with channel-up-modulating cAMP solution. 0.01-1 microM FSK had no effect on up-modulated currents, whereas high FSK rapidly and reversibly inhibited ICa,L by < or = 42% without affecting ICl. High FSK also inhibited ICa,L in myocytes dialyzed with protein kinase A inhibitor. External but not internal application of the inactive analog 1,9-dideoxy-FSK (30-100 microM) inhibited basal ICa,L. The inhibition was dependent on holding potential and involved a speeding up of ICa,L inactivation and a slowing of recovery from inactivation. We conclude that FSK inhibits cardiac ICa,L by reducing the availability of Ca2+ channels.