A new therapeutic approach to treat psoriasis by inhibition of fatty acid oxidation by Etomoxir.

BACKGROUND: The dogma in psoriasis is that due to pathogen-induced inflammatory responses, an autoreactive immune response is induced that leads to tissue destruction. However, this model might be too simplistic. Literature data suggest that the expression of enzymes crucial for fatty acid oxidation is upregulated in the skin of patients with psoriasis compared with healthy individuals. OBJECTIVES: To examine the influence of fatty acid oxidation on psoriasis with regard to expression and activity of the key enzyme in fatty acid oxidation, carnitine palmitoyltransferase-1 (CPT-1) and the effect of the CPT-1 inhibitor, Etomoxir. METHODS: Experiments were performed with homogenates of lesional and healthy skin, fibroblast cultures and a model of human psoriatic skin transplanted on immune-deficient BNX mice. RESULTS: CPT-1 was highly active in lesional skin. Etomoxir was able to block CPT-1 activity in skin, implying that this antagonist may have the potential to suppress psoriasis when administered topically. In the mouse model, Etomoxir had an antipsoriatic effect that was at least as good as that of betamethasone, as evidenced by reduction of epidermal thickness, keratinocyte proliferation and differentiation. CONCLUSIONS: We conclude that fatty acid metabolism and in particular CPT-1 may be an excellent target for treatment of psoriasis.

The tandem affinity purification (TAP) method: a general procedure of protein complex purification.

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level. Prior knowledge of complex composition or function is not required. The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented. The TAP method was initially developed in yeast but can be successfully adapted to various organisms. Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration.

Partial purification of the yeast U2 snRNP reveals a novel yeast pre-mRNA splicing factor required for pre-spliceosome assembly.

We have partially purified the U2 snRNP of Saccharomyces cerevisiae. Identification of some proteins consistently found in the purified fractions by nanoelectrospray mass spectrometry indicated the presence of a novel splicing factor named Rse1p. The RSE1 gene is essential and codes for a 148.2 kDa protein. We demonstrated that Rse1p associates specifically with U2 snRNA at low salt concentrations. In addition, we showed that Rse1p is a component of the pre-spliceosome. Depletion of Rse1p and analysis of a conditional mutant indicated that Rse1p was required for efficient splicing in vivo. In vitro Rse1p is required for the formation of pre-spliceosomes. Database searches revealed that Rse1p is conserved in humans and that it belongs to a large protein family that includes polyadenylation factors and DNA repair proteins. The characteristics of Rse1p suggest that its human homologue could be a subunit of the SF3 splicing factor.

The yeast U2A'/U2B complex is required for pre-spliceosome formation.

Human U2 snRNP contains two specific proteins, U2A' and U2B", that interact with U2 snRNA stem-loop IV. In Saccharomyces cerevisiae, only the counterpart of human U2B", Yib9p, has been identified. Database searches revealed a gene potentially coding for a protein with striking similarities to human U2A', henceforth called LEA1 (looks exceptionally like U2A'). We demonstrate that Lea1p is a specific component of the yeast U2 snRNP. In addition, we show that Lea1p interacts directly with Yib9p. In vivo association of Lea1p with U2 snRNA requires Yib9p. Reciprocally, Yib9p binds to the U2 snRNA only in the presence of Lea1p in vivo, even though it has been previously shown to associate on its own with the U2 snRNA stem-loop IV in vitro. Strains lacking LEA1 and/or YIB9 grow slowly, are temperature sensitive and contain reduced levels of U2 snRNA. Pre-mRNA splicing is strongly impaired in these cells. In vitro studies demonstrate that spliceosome assembly is blocked prior to addition of U2 snRNP. This phenotype can be rescued partially, but specifically, by addition of the corresponding recombinant protein(s). This demonstrates a specific role for the yeast U2 snRNP specific proteins during formation of the pre-spliceosome.

Constitutive and carbon source-responsive promoter elements are involved in the regulated expression of the Saccharomyces cerevisiae malate synthase gene MLS1.

The malate synthase gene, MLS1, of the yeast Saccharomyces cerevisiae is transcriptionally regulated by the carbon source in the growth medium. A MLS1-lacZ fusion gene, expressed at a basal level in the presence of 2% glucose, is derepressed more than 100-fold under conditions of sugar limitation. No evidence for MLS1 induction by oleic acid was found. By deletion analysis of the MLS1 control region, we identified two sites, UAS1 and UAS2, as important for efficient derepression of the gene. Both sites contain sequences that resemble the previously characterized carbon source-responsive element (CSRE) found in the promoter of the isocitrate lyase gene ICL1. Indeed, UAS1 and UAS2 in the MLS1 upstream region turn out to be functional CSRE sequence variants. This finding allowed us to define a modified version of the CSRE consensus sequence (CCRTYSRNCCG). Protein binding to UAS1MLS1 was observed with extracts from derepressed but not from repressed cells, and could be competed for by an excess of the unlabelled CSRE (ICL1) sequence. No competition was observed with a mutated CSRE variant. Site-directed mutagenesis of both CSREs in the MLS1 promoter reduced gene activation under derepressing conditions to 20% of the wild-type level. The same decrease was observed with the wild-type MLS1 promoter in a cat8 mutant, lacking an activator of CSRE-dependent transcription. The CSRE/Cat8p-independent activation of MLS1 is mediated by constitutive UAS elements. The pleiotropic transcription factor Abf1p, which binds to the MLS1 upstream region, may contribute to constitutive activation. Thus, in order to ensure the severe glucose repression of MLS1 observed, repressor elements that respond to the carbon source must counteract constitutive activation. In summary, ICL1 and MLS1 share common cis-acting elements, although a distinct mechanism of carbon source control also contributes to MLS1 regulation.

[5-years of experience with tranquilizing-analgesics in laryngo-tracheo-bronchoscopic procedures in newborns and infants ]. / 5-Jahres-Erfahrungen mit der Tranquanalgesie bei laryngo-tracheo-bronchoskopischen Eingriffen im Neugeborenen- und Säuglingsalter.

Tranquilizing-analgesics (diazepam-ketamine combination anaesthesia) and O2-injector respiration has proved during a period of 5 years in 270 laryngo-tracheo-bronchoscopies of newborns and infants to be an outstandingly well-controllable anaesthesia applicable by the intravenous route which could be applied without danger of depression of breathing or circulation and without injury of the laryngeal protection reflex in this early period of life by means of a balanced cooperation between operator and anaesthesiologist. No serious complications have been observed. The authors' own experiences in handling of the method are described.

Effect of atropine on pancreatic response to HCl and secretin.

In dogs with gastric and pancreatic fistulas, we studied the effect of atropine on the pancreatic secretory response to secretin and intestinal HCl. Atropine sulfate (20 micrograms.kg-1.h-1 iv) significantly depressed basal bicarbonate and protein output. Atropine depressed bicarbonate responses to low doses (62.5, 125, 250, and 500 ng.kg-1.h-1) of secretin but had no significant effect on responses to high doses (1,000 and 2,000 ng.kg-1.h-1). Secretin, with or without atropine, did not stimulate pancreatic protein output above basal. Atropine depressed bicarbonate responses to low loads (3, 6, and 12 mmol.h-1) of HCl but had no significant effect on responses to high loads (12, 24, and 48 mmol.h-1). Intraduodenal HCl produced a dose-dependent increase in protein output. Atropine abolished protein responses to low loads (3 and 6 mmol.h-1) but did not affect responses to high loads (24 and 48 mmol.h-1) of HCl. These findings are compatible with the hypotheses that a) endogenous cholinergic activity augments the pancreatic bicarbonate response to secretin, and b) the pancreatic protein response to intraduodenal HCl is, at least in part, mediated cholinergically.