[Management and prognosis of patients with squamous cell carcinomas of the nasal cavity and the paranasal sinuses]. / Management und Prognose von Patienten mit Plattenepithelkarzinomen der Nasenhaupt- und Nasennebenhöhlen.

Squamous cell carcinomas (SCC) of the nasal cavity and the paranasal sinuses are a very rare and poorly understood tumor entity. To date, no consistent management strategy exists. The purpose of our study was to demonstrate our therapeutic strategy and to correlate clinicopathological features with clinical follow-up data.45 patients with primarily resected SCC of the nasal cavity (n=35) and the paranasal sinuses (n=10) between 1994 and 2010 were reviewed retrospectively (mean follow-up period 2.6 years; range 0.3 to 14.9 years).Tumors of the nasal cavity were diagnosed at an early stage (97% T1 and T2) whereas tumors of the parasinuses were found at an advanced stage (90% T3 and T4). Lymph node metastases were only found 2 patients. 13 patients (29%) had a local tumor progress, 2 patients showed lymph node metastases and 4 patients had distant metastases in follow up. The prognosis of tumors of the nasal cavitiy or the paranasal sinuses was bad (31% 5-year overall survival) especially by patients with a relapse.Reconstructive surgery was performed after 12 months, when early local relapse could be excluded. There was no positive correlation between clinicopathological features and survival data.The prognosis of tumors of the nasal cavity and paranasal sinuses depends mainly on the control of local tumor growth. Modern strategies of surgical treatment in combination with radiotherapy need to be implemented in an effort to achieve continuous tumor-free survival.

[Grisel's syndrome after otoplasty]. / Grisel-Syndrom nach Anthelixplastik.

Grisel's syndrome is known as a very rare complication of ENT surgery. It is described as non-traumatic atlantoaxial rotatory subluxation, often seen after tonsillectomy or adenoidectomy in children. Therapy is staged according to the Fielding classification. We report the case of a 9-year-old female patient with Grisel's syndrome after otoplasty. The diagnosis was confirmed by CT scan. Manual reposition was performed under general anaesthesia, followed by temporary immobilization with a Minerva orthesis.

The C-terminus of p63 contains multiple regulatory elements with different functions.

The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ∼13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63.

Role of molecular phonons and interfacial-temperature discontinuities in water evaporation.

During steady-state water evaporation, when the vapor phase is heated electrically, the temperature on the vapor side of the interface has been reported to be as much as 27.83 degrees C greater than that on the liquid side. The reported interfacial temperatures were measured with thermocouple beads that were less than 50 microm in diameter and centered 35 microm from the interface in each phase. We examine the reliability of these measurements by using them with a theory of kinetics to predict the interfacial-liquid temperature. The predicted temperature discontinuities are found to be in agreement with those measured up to a temperature discontinuity of 15.69 degrees C , but larger discontinuities cannot be confirmed because of uncertainties in the vapor-phase pressure measurements. The theory of kinetics used in the analysis includes molecular phonons in the expression for the evaporation flux. We show it is essential to include these terms if the theory is to be used to predict the temperature discontinuities.

[Repeated dual-probe pH monitoring as a diagnostic tool to control therapy for laryngopharyngeal reflux]. / Therapiekontrolle bei laryngopharyngealem Reflux durch wiederholte 2-Kanal-pH-Metrie.

OBJECTIVES: The most effective current treatment option for patients suffering from laryngopharyngeal reflux (LPR) is the use of proton pump inhibitors (PPIs). Compared to other PPIs, esomeprazole seems to provide best 24-h control of intragastric acid. However, some patients remain resistant to medical acid suppression with PPIs. The aim of this investigation was to identify the number of non-responders among patients suffering from LPR being treated by esomeprazole 40 mg once daily (officially approved maximum dosage). PATIENTS AND METHODS: Between June 2004 and January 2006, 27 patients suffering from LPR diagnosed by dual-probe pH monitoring were treated with esomeprazole 40 mg once daily. After 13-54 days (mean 28 days) while still under PPI-treatment, the 24-h pH-study was repeated in order to control the effectiveness of therapy. Patients with at least a reduction of the reflux area index (RAI) compared to the result before treatment were categorized as responders. Furthermore, the number of patients with a measurable reduction of proximal reflux episodes under PPI-treatment was quantified. RESULTS: Repeated pH monitoring during PPI therapy revealed a reduction of the RAI in 22 of 27 patients. Five patients, however, showed a higher RAI despite medical treatment (19%). In 13 patients (48%), treatment with 40 mg esomeprazole once daily reduced the RAI to a normal value (<6.3). In 18 of 27 patients, the number of proximal reflux episodes has decreased (67%). CONCLUSION: In a number of patients suffering from LPR, treatment with esomeprazole 40 mg once daily did not provide any measurable proximal acid reduction. Repeated pH monitoring during treatment is an adequate diagnostic tool to control the therapeutic effect of PPIs objectively and to identify non-responders at an early point.

Thermocapillary transport of energy during water evaporation.

When evaporation occurs at a spherical water-vapor interface maintained at the circular mouth of a small funnel, studies of the energy transport have indicated that thermal conduction alone does not provide enough energy to evaporate the liquid at the observed rate. If the Gibbs model of the interface is adopted and the "surface-thermal capacity" is assigned a value of 30.6+/-0.8 kJ/(m2 K), then for evaporation experiments with the interfacial temperature in the range -10 degrees C< or =TLV< or =3.5 degrees C and Marangoni number (Ma) in the range 100

Bi-directional flow sensor with a wide dynamic range for medical applications.

This paper describes a novel three-wire thermal flow sensor for medical applications. The present innovation for low-frequency measurements involves the use of a pulsed-wire anemometer with a comparatively large wire diameter (12.5 microm and larger) together with a novel signal processing approach. A small wire is heated using a sinusoidal alternating current, and two sensing wires, acting as resistance thermometers, are set parallel to, and at a small distance on either side of, the pulsed wire. The thermal wake of the pulsed wire is convected downstream to one of the two receiving wires which detect its delayed arrival. This arrangement allows the sensing of both the direction and the flow velocity component normal to the three probes. By appropriate signal processing, the present sensor can be operated such that the phase shift between the periodic current that drives the central wire and the detected signal by either the upstream or downstream wire takes into account a combination of convection, diffusion and the finite thermal response time of both the pulsed wire and the receiving wire. Because the time constants increase as the flow velocity decreases, the time lag due to thermal inertia supplements the time lag due to the true time of flight, thus yielding an effective operating range of 0.05 m/s

Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omega -hydroxylation in development.

We describe lacerata (lcr) mutants of Arabidopsis, which display various developmental abnormalities, including postgenital organ fusions, and report cloning of the LCR gene by using the maize transposon Enhancer/Suppressor-mutator (En/Spm). The pleiotropic mutant phenotype could be rescued by genetic complementation of lcr mutants with the wild-type LCR gene. The LCR gene encodes a cytochrome P450 monooxygenase, CYP86A8, which catalyzes omega-hydroxylation of fatty acids ranging from C12 to C18:1, as demonstrated by expression of the gene in yeast. Although palmitic and oleic acids were efficient substrates for LCR, 9,10-epoxystearate was not metabolized. Taken together with previous studies, our findings indicate that LCR-dependent omega-hydroxylation of fatty acids could be implicated in the biosynthesis of cutin in the epidermis and in preventing postgenital organ fusions. Strikingly, the same pathway seems to control trichome differentiation, the establishment of apical dominance, and senescence in plants.

Localization of CYP86B1 in the outer envelope of chloroplasts.

CYP86B1 was cloned from a cDNA library and the protein expressed in E. coli. The protein gave the expected carbon monoxide difference spectrum. Using in vitro import assays with isolated pea chloroplasts, CYP86B1 was shown to be associated with the outer chloroplastic envelope membrane. This study provides the first direct evidence for a chloroplast-localized cytochrome P450-dependent monooxygenase.

A conservative amino acid substitution alters the regiospecificity of CYP94A2, a fatty acid hydroxylase from the plant Vicia sativa.

Fatty acid omega-hydroxylation is involved in the biosynthesis of the plant cuticle, formation of plant defense signaling molecules, and possibly in the rapid catabolism of free fatty acids liberated under stress conditions. CYP94A2 is a cytochrome P450-dependent medium-chain fatty acid hydroxylase that was recently isolated from Vicia sativa. Contrary to CYP94A1 and CYP86A1, two other fatty acid hydroxylases previously characterized in V. sativa and Arabidopsis thaliana, CYP94A2 is not a strict omega-hydroxylase, but exhibits chain-length-dependent regioselectivity of oxidative attack. Sequence alignments of CYP94A2 with CYP94A1 and molecular modeling studies suggested that F494, located in SRS-6 (substrate recognition site) was involved in substrate recognition and positioning. Indeed, a conservative amino acid substitution at that position markedly altered the regiospecificity of CYP94A2. The observed shift from omega toward omega-1 hydroxylation was prominent with lauric acid as substrate and declined with increasing fatty acid chain length.

Reynolds stress under a change of frame of reference.

In this paper, we study the characteristics of the Reynolds stress under a change of frame, as defined by the Euclidean group of transformation. We show that being subject to the dynamical processes induced from the mean Navier-Stokes equations, the invariance group of the fluctuating velocity and the Reynolds stress is no longer the Euclidean group of transformation, which is merely a kinematical aspect, but reduces to the extended Galilean group of transformation. As a consequence, in contrast to developing the constitutive equations for the Cauchy stress in continuum mechanics, wherein the principle of material frame-indifference is a guiding principle, the frame-dependent kinematical quantities, e.g., the mean spin tensor, may be allowed to play an effective role as the constitutive variable in turbulence modeling.

CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the omega-alcohol and to the corresponding diacid.

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 +/- 0.2 microM and Vmax,app = 27 +/- 1 nmol x min(-1) x nmol(-1) P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10-epoxy-octadecan-1,18-dioic acid. The diacid was also produced in microsomal incubations of 18-hydroxy-9,10-epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the omega-hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.

omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defence.

The C(18) fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa. The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S,10R and 9R,10S enantiomers were incubated separately. We determined respective K(m) and V(max) values of 1.2+/-0.1 microM and 19.2+/-0.3 nmol/min per nmol of cytochrome P450 for the 9R,10S enantiomer and of 5.9+/-0.1 microM and 20.2+/-1.0 nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially formed in V. sativa microsomes. Cutin analysis of V. sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C(16) fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.

Production in vitro by the cytochrome P450 CYP94A1 of major C18 cutin monomers and potential messengers in plant-pathogen interactions: enantioselectivity studies.

The major C(18) cutin monomers are 18-hydroxy-9,10-epoxystearic and 9,10,18-trihydroxystearic acids. These compounds are also known messengers in plant-pathogen interactions. We have previously shown that their common precursor 9,10-epoxystearic acid was formed by the epoxidation of oleic acid in Vicia sativa microsomes (Pinot, Salaün, Bosch, Lesot, Mioskowski and Durst (1992) Biochem. Biophys. Res. Commun. 184, 183-193). Here we determine the chirality of the epoxide produced as (9R,10S) and (9S,10R) in the ratio 90:10 respectively. We further show that microsomes from yeast expressing the cytochrome P450 CYP94A1 are capable of hydroxylating the methyl terminus of 9,10-epoxystearic and 9,10-dihydroxystearic acids in the presence of NADPH to form the corresponding 18-hydroxy derivatives. The reactions were not catalysed by microsomes from yeast transformed with a void plasmid or in absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid with microsomes of yeast expressing CYP94A1, the chirality of the residual epoxide was shifted to 66:34 in favour of the (9S,10R) enantiomer. Both enantiomers were incubated separately and V(max)/K(m) values of 16 and 3.42 ml/min per nmol of P450 for (9R, 10S) and (9S,10R) respectively were determined, demonstrating that CYP94A1 is enantioselective for the (9R,10S) enantiomer, which is preferentially formed in V. sativa microsomes. Compared with the epoxide, the diol 9,10-dihydroxystearic acid was a much poorer substrate for the omega-hydroxylase, with a measured V(max)/K(m) of 0.33 ml/min per nmol of P450. Our results indicate that the activity of CYP94A1 is strongly influenced by the stereochemistry of the 9, 10-epoxide and the nature of substituents on carbons 9 and 10, with V(max)/K(m) values for epoxide>>oleic acid>diol.

Cloning and functional characterization of CYP94A2, a medium chain fatty acid hydroxylase from Vicia sativa.

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A2) was isolated from a Vicia sativa library. CYP94A2 displays 58% sequence identity with CYP94A1, a fatty acid omega-hydroxylase isolated from the same material. Heterologous expression of CYP94A2 in Saccharomyces cerevisiae yeast strain WAT11 shows that it catalyses the hydroxylation of myristic (C14) acid with a K(m(app)) of 4.0 microM and a turnover rate number of 80 min(-1). In addition, lauric (C12) and palmitic (C16) acids were hydroxylated at a ten-fold lower rate, while C18 fatty acids were not oxidized. Remarkably, the regiospecificity of hydroxylation is different for the C12, C14, and C16 fatty acids and appears to be correlated with the length of the carbon chain. Northern blot analysis showed a low level of constitutive expression of CYP94A2 in V. sativa seedlings. In contrast to CYP94A1, transcript accumulation of CYP94A2 was only weakly enhanced in seedlings treated with clofibrate or methyl jasmonate, indicating that both substrate range and gene regulation of the two fatty acid hydroxylases are different.

Methyl jasmonate induces lauric acid omega-hydroxylase activity and accumulation of CYP94A1 transcripts but does not affect epoxide hydrolase activities in vicia sativa seedlings

Treatment of etiolated Vicia sativa seedlings by the plant hormone methyl jasmonate (MetJA) led to an increase of cytochrome P450 content. Seedlings that were treated for 48 h in a 1 mM solution of MetJA stimulated omega-hydroxylation of 12:0 (lauric acid) 14-fold compared with the control (153 versus 11 pmol min-1 mg-1 protein, respectively). Induction was dose dependent. The increase of activity (2.7-fold) was already detectable after 3 h of treatment. Activity increased as a function of time and reached a steady level after 24 h. Northern-blot analysis revealed that the transcripts coding for CYP94A1, a fatty acid omega-hydroxylase, had already accumulated after 1 h of exposure to MetJA and was maximal between 3 and 6 h. Under the same conditions, a study of the enzymatic hydrolysis of 9,10-epoxystearic acid showed that both microsomal and soluble epoxide hydrolase activities were not affected by MetJA treatment.

Molecular cloning and functional expression in yeast of CYP76B1, a xenobiotic-inducible 7-ethoxycoumarin O-de-ethylase from Helianthus tuberosus.

In order to obtain plant markers of chemical stress and possible tools for the bio-monitoring of pollution, a protein purification/PCR approach was used to isolate cDNAs of xenobiotic-inducible P450 oxygenases. O-dealkylation of 7-ethoxycoumarin is catalysed in Helianthus tuberosus by cytochromes P450 strongly inducible by a wide range of xenobiotics. Therefore, a 7-ethoxycoumarin O-de-ethylase (ECOD) was purified from induced tuber tissues (Batard et al., 1995). A primer designed from an internal peptide sequence, but also corresponding to a conserved P450 haem-binding region, led to the generation of a gene-specific probe corresponding to a P450 strongly inducible by aminopyrine. Two partial and 98% identical coding sequences were isolated from a cDNA library prepared from aminopyrine-induced tuber. A full-length cDNA was reconstituted by 5'-RACE elongation. The protein deduced from this full-length sequence, with 41.1% amino acid identity to CYP76A1 and high phylogenetic relationship to other CYP76s, was termed CYP76B1. CYP76B1 was expressed in yeast. Microsomes from the transformed yeast catalysed the NADPH-dependent O-dealkylation of 7-ethoxycoumarin. However, protein sequence as well as enzymological data indicated that CYP76B1 does not correspond to the purified ECOD protein. These results confirm previous data and demonstrate that several P450s in H. tuberosus are capable of actively catalysing the O-de-ethylation of ethoxycoumarin. Determination of the steady-state level of CYP76B1 transcripts after slicing tuber tissues and ageing them in water, alone or in the presence of various chemicals, showed that the expression of this P450 was not responsive to mechanical stress, but was strongly induced by chemical treatments. CYP76B1 thus appears to be a good potential marker of chemical stress and of environmental pollution.

Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis.

The chemical tagging of a cytochrome P-450-dependent lauric acid omega-hydroxylase from clofibrate-treated Vicia sativa seedlings with [1-14C]11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1. We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana. The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1. The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1. Both E/Z and Z/E configurations of 9, 12-octadecadienoic acids were omega-hydroxylated. Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1). The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized. Similar to animal omega-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment. Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug. The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.

Cloning, expression in yeast, and functional characterization of CYP81B1, a plant cytochrome P450 that catalyzes in-chain hydroxylation of fatty acids.

Several omega and in-chain fatty acid hydroxylases have been characterized in higher plants. In microsomes from Helianthus tuberosus tuber the omega-2, omega-3, and omega-4 hydroxylation of lauric acid is catalyzed by one or a few closely related aminopyrine- and MnCl2-inducible cytochrome P450(s). To isolate the cDNA and determine the sequences of the(se) enzyme(s), we used antibodies directed against a P450-enriched fraction purified from Mn2+-induced tissues. Screening of a cDNA expression library from aminopyrine-treated tubers led to the identification of a cDNA (CYP81B1) corresponding to a transcript induced by aminopyrine. CYP81B1 was expressed in yeast. A systematic exploration of its function revealed that it specifically catalyzes the hydroxylation of medium chain saturated fatty acids, capric (C10:0), lauric (C12:0), and myristic (C14:0) acids. The same metabolites were obtained with transgenic yeast and plant microsomes, a mixture of omega-1 to omega-5 monohydroxylated products. The three fatty acids were metabolized with high and similar efficiencies, the major position of attack depending on chain length. When lauric acid was the substrate, turnover was 30.7 +/- 1.4 min-1 and Km(app) 788 +/- 400 nM. No metabolism of long chain fatty acids, aromatic molecules, or herbicides was detected. This new fatty acid hydroxylase is typical from higher plants and differs from those already isolated from other living organisms.

CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase.

The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.