Evaluation of the potential stranding risk for aquatic organisms according to long-term morphological changes and grain size in alpine rivers impacted by hydropeaking.

Hydropeaking is one of the major hydropower-related disturbances of natural processes in river systems. The artificial flow fluctuations that are caused by the on-demand production of electricity are known for their severe impacts on aquatic ecosystems. These particularly affect those species and life stages that are not able to adjust their habitat selection to rapid up- and downramping rates. To date, the stranding risk has both experimentally and numerically mainly been investigated with variable hydropeaking graphs over stable river bathymetries. There is a lack of knowledge on how single, discrete peaking events vary concerning their impact on the stranding risk when the river morphology changes in the long-term perspective. The present study precisely addresses this knowledge gap by investigating morphological changes on the reach scale over a period of 20 years and the related variability of the lateral ramping velocity as a proxy for stranding risk. Two alpine gravel bed rivers impacted by hydropeaking over decades were tested by applying a one-dimensional and two-dimensional unsteady modelling approach. Both the Bregenzerach River and the Inn River exhibit alternating gravel bars on the reach scale. The results of the morphological development, however, showed different developments in the period 1995-2015. The Bregenzerach River displayed continuous aggradation (uplift of river bed) over the various selected submonitoring periods. In contrast, the Inn River showed continuous incision (erosion of river bed). The stranding risk exhibited high variability on a single cross-sectional basis. However, on the reach scale, no significant changes in stranding risk were calculated for either river reach. In addition, the impacts of river incision on the substrate composition were investigated. Here, in line with preceding studies, the results show that the coarsening of substrate increases the stranding risk and that especially the d90 (90 % finer of the grain size distribution) must be considered. The present study reveals that the quantified stranding risk of aquatic organisms is a function of the general morphological (bar) characteristics of the impacted river and both the morphological and grain size development have an impact on the potential stranding risk of aquatic organisms and should be considered in the revision of licences in the management of multi-stressed river systems.

Aquatic pathogens and biofouling: pilot study of ostreid herpesvirus 1 translocation by bivalves.

Ostreid herpesvirus 1 (OsHV-1) has caused mass mortalities in Pacific oysters (Crassostrea gigas) in Europe, Australia, and New Zealand. While aquaculture-associated movements of infected Pacific oysters are a well-known cause of OsHV-1 spread once established in a region, translocation via biofouling of aquaculture equipment or vessels needs further investigation to explain the more distant spread of OsHV-1. Laboratory experiments were designed to test for transmission of OsHV-1 between infected and naïve Pacific oysters via a simulated biofouling translocation scenario. Three common biofouling species [Sydney rock oysters (Saccostrea glomerata), Mediterranean mussels (Mytilus galloprovincialis) and Pacific oysters] were tested as intermediaries using a cohabitation challenge with Pacific oysters infected by injection. Transmission occurred, albeit for one of eight replicates when Pacific oysters were the intermediary species. This demonstrated a possible pathway for pathogen spread via biofouling containing Pacific oysters while highlighting the complexity of OsHV-1 transmission. Such complexities require further investigation to inform future risk assessments and management of fouled aquaculture equipment and vessels.

L'impact des microvariants du virus herpétique ostreid herpesvirus 1 sur la culture d'huîtres creuses dans les hémisphères Nord et Sud depuis 2008.

Mollusc farming is the third most productive aquaculture activity in the world, and the Pacific oyster (Crassostrea gigas) is one of the most important farmed species. Since 2008, mass mortalities in C. gigas due to ostreid herpesvirus 1 microvariants have challenged the viability of this industry in Europe, New Zealand and Australia. Ten years after the emergence of this disease, there is evidence that the industry has become consolidated into fewer, larger companies, with the displacement of small farming enterprises and loss of employment in coastal communities. Rather than seeking technical solutions, the industry has turned to compensatory production strategies, such as increasing the number of spat placed on farms, higher market prices for table oysters and direct marketing, which appear to have allowed profitability. Biosecurity policies and responses to outbreaks, including those from within the industry, have had unintended consequences for hatcheries and farmers in areas free of disease, mainly caused by restrictions on animal movements, and have not prevented global spread. There may be opportunities for better coordination of industry and government responses to epizootic disease emergence in aquaculture. There is certainly a need for increased adoption of technical advances from research, once these solutions have been adequately verified.

L'élevage de mollusques occupe le troisième rang mondial parmi les activités de l'aquaculture en termes de production ; l'une des principales espèces élevées est l'huître creuse (Crassostrea gigas). Depuis 2008, la rentabilité des élevages de C. gigas en Europe, en Nouvelle-Zélande et en Australie a été fortement compromise par une mortalité massive due à des microvariants du virus herpétique Ostreid herpesvirus 1. Dix ans après l'émergence de cette maladie, on observe une forte concentration du secteur autour d'entreprises moins nombreuses mais de plus grande envergure qui ont remplacé l'ancien tissu d'exploitations artisanales et occasionné un déclin de l'emploi dans les communautés littorales. Au lieu de rechercher des solutions techniques, le secteur a eu recours à des stratégies de compensation axées sur la production, par exemple en augmentant le nombre de naissains mis en place dans les fermes, en augmentant le prix des huîtres de consommation ou en développant la vente directe, stratégies dont l'impact sur la rentabilité semble avoir été positif. En revanche, les mesures de biosécurité mises en place et les réponses apportées aux foyers, y compris celles introduites par le secteur lui-même ont eu des conséquences imprévues pour les écloseries et les éleveurs des zones indemnes de maladie, principalement en raison des restrictions imposées aux transferts d'animaux, sans pour autant prévenir la propagation de la maladie à l'échelle mondiale. Une meilleure coordination des réponses sectorielles et publiques face à l'émergence des maladies épizootiques affectant l'aquaculture devrait être possible. Il sera également indispensable de recourir davantage aux avancées techniques mises au point par la recherche dès que ces solutions auront été dûment validées.

La producción de moluscos es la tercera actividad acuícola más productiva del mundo, y la ostra japonesa (o del Pacífico) (Crassostrea gigas) ocupa un lugar destacado entre las principales especies cultivadas. Desde 2008, la viabilidad de esta industria en Europa, Nueva Zelanda y Australia está amenazada por episodios de mortandad masiva de C. gigas causados por microvariantes del herpesvirus de los ostreidos 1 (ostreid herpesvirus 1). Diez años después de la aparición de la enfermedad, lo que se observa es que la industria se ha ido concentrando en unas pocas empresas de grandes dimensiones, que han desplazado a las pequeñas empresas ostrícolas y causado la pérdida de numerosos empleos en las comunidades costeras. En lugar de buscar soluciones técnicas, la industria ha optado más bien por estrategias de producción compensatorias (como aumentar el número de semillas de ostra por explotación, subir los precios de mercado de las ostras de mesa o recurrir a la comercialización directa) que parecen haber deparado rentabilidad. Las políticas de seguridad biológica y la respuesta a los brotes, incluida la del propio sector, han tenido consecuencias imprevistas para los viveros y acuicultores situados en zonas libres de la enfermedad, debido sobre todo a las restricciones impuestas a los desplazamientos de animales, sin que ello haya servido para impedir la diseminación mundial de esta patología. Puede haber margen para coordinar más eficazmente las respectivas respuestas de la industria y de los poderes públicos ante la aparición de enfermedades epizoóticas en la acuicultura. Lo que sin ninguna duda es necesario es incorporar en mayor medida los adelantos técnicos resultantes de la investigación, una vez contrastada debidamente cada solución.

Characterization of proliferative effects of insulin, insulin analogues and insulin-like growth factor-1 (IGF-1) in human lung fibroblasts.

Insulin has been approved for inhaled application, but safety concerns remain, because of un-physiologically high insulin concentrations in the lung. Since insulin may act as growth factor, possible proliferative effects of insulin, insulin analogues and insulin-like growth factor-1 (IGF-1) on human lung fibroblasts were studied. As measure of proliferation [(3)H]-thymidine incorporation was studied in HEL-299, MRC-5, IMR-90 and primary human lung fibroblasts. In all cells, mRNA encoding IGF-1 receptors and two variants of insulin receptors was detected. Insulin and IGF-1 stimulated [(3)H]-thymidine incorporation in all cells. Comparison of the concentration-dependent effects in HEL-299 cells showed that IGF-1 and insulin glargine were more potent (EC(50), 3 and 6 nM) and more effective (maximum increase, by 135-150%) than insulin and insulin detemir (EC(50), 22 and 110 nM; maximum increase: by 80%). Proliferative effects of IGF-1 and insulin were inhibited to the same extent by an antibody (1H7) directed against the IGF-1 receptor α-subunit. Insulin-induced stimulation of [(3)H]-thymidine incorporation was reduced by 83% after siRNA-mediated down-regulation of IGF-1 receptor by about 75%, but not affected by a similar down-regulation of the insulin receptor. Insulin and IGF-1 caused rapid up-regulation of the early genes FOS, EGR-1 and EGR-2 as well as of the gene coding for IGF-1. In conclusion, in human lung fibroblasts insulin exerts marked proliferative effects and the pharmacological profile of this response as well as specific receptor knock-down experiments suggest mediation via IGF-1 receptors. The risk of unwanted structural lung alterations by long-term inhalative application of insulin should be considered.

Spectroscopic characterization of flavin mononucleotide bound to the LOV1 domain of Phot1 from Chlamydomonas reinhardtii.

The absorption and emission behavior of flavin mononucleotide (FMN) in the light-, oxygen- and voltage-sensitive (LOV) domain LOV1 of the photoreceptor Phot1 from the green alga Chlamydomonas reinhardtii was studied. The results from the wild-type (LOV1-WT) were compared with those from a mutant in which cysteine 57 was replaced by serine (LOV1-C57S), and with free FMN in aqueous solution. A fluorescence quantum yield of phi(F) = 0.30 and a fluorescence lifetime of tau(F) = 4.6 ns were determined for FMN in the mutant LOV1-C57S, whereas these quantities are reduced to about phi(F) = 0.17 and tau(F) = 2.9 ns for LOV1-WT, indicating an enhanced intersystem crossing in LOV1-WT because of the adjacent sulfur of C57. A single-exponential fluorescence decay was observed in picosecond laser time-resolved fluorescence measurements for both LOV1-WT and LOV1-C57S as expected for excited singlet state relaxation by intersystem crossing and internal conversion. An excitation intensity dependent fluorescence signal saturation was observed in steady-state fluorescence measurements for LOV1-WT, which is thought to be because of the formation of a long-lived intermediate flavin-C(4a)-cysteinyl adduct in the triplet state (few microseconds triplet lifetime, adduct lifetime around 150 s). No photobleaching was observed for LOV1-C57S, because no thiol group is present in the vicinity of FMN for an adduct formation.

Sulfur polymer solidification/stabilization of elemental mercury waste.

Elemental mercury, contaminated with radionuclides, presents a waste disposal problem throughout the Department of Energy complex. In this paper we describe a new process to immobilize elemental mercury wastes, including those contaminated with radionuclides, in a form that is non-dispersible, will meet EPA leaching criteria, and has low mercury vapor pressure. In this stabilization and solidification process, elemental mercury is combined with an excess of powdered sulfur polymer cement (SPC) and sulfide additives in a mixing vessel and heated to approximately 40 degrees C for several hours, until all of the mercury is converted into mercuric sulfide (HgS). Additional SPC is then added and the temperature of the mixture raised to 135 degrees C, resulting in a molten liquid which is poured into a mold where it cools and solidifies. The final treated waste was characterized by powder X-ray diffraction and found to be a mixture of the hexagonal and orthorhombic forms of mercuric sulfide. The Toxicity Characteristic Leaching Procedure was used to assess mercury releases, which for the optimized process averaged 25.8 microg/l, with some samples being well below the new EPA Universal Treatment Standard of 25 microg/l. Longer term leach tests were also conducted, indicating that the leaching process was dominated by diffusion. Values for the effective diffusion coefficient averaged 7.6x10(-18) cm2/s. Concentrations of mercury vapor from treated waste in equilibrium static headspace tests averaged 0.6 mg/m3.

Human and rat alveolar macrophages express multiple EDG receptors.

Endothelial differentiation gene (EDG) receptors are a new family of eight G protein-coupled receptors for the lysophospholipids lysophosphatitic acid and sphingosine-1-phosphate. In the present experiments, the expression of EDG receptors in rat and human alveolar macrophages was studied by reverse transcription-polymerase chain reaction (RT-PCR). In alveolar macrophages of both species, mRNA for multiple EDG receptors could be detected, but the pattern of expression was different in both species. In human alveolar macrophages, mRNA for EDG1, EDG2, EDG4, EDG7 receptors and, to a lesser extent, for the EDG7 receptor was detected, whereas in rat macrophages, mRNA for EDG2, EDG5 receptors and, to a lesser extent, for the EDG6 receptor was found. In functional experiments, it was observed that lysophosphatitic acid and sphingosine-1-phosphate can stimulate O(2)(-) generation in rat and human alveolar macrophages suggesting that lysophosphatitic acid and sphingosine-1-phosphate possibly acting via EDG receptors may play a role in controlling the activation of macrophages.

The abundant retinal protein of the Chlamydomonas eye is not the photoreceptor for phototaxis and photophobic responses.

The chlamyopsin gene (cop) encodes the most abundant eyespot protein in the unicellular green alga Chlamydomonas reinhardtii. This opsin-related protein (COP) binds retinal and was thought to be the photoreceptor controlling photomovement responses via a set of photoreceptor currents. Unfortunately, opsin-deficient mutants are not available and targeted disruption of non-selectable nuclear genes is not yet possible in any green alga. Here we show that intron-containing gene fragments directly linked to their intron-less antisense counterpart provide efficient post-transcriptional gene silencing (PTGS) in C. reinhardtii, thus allowing an efficient reduction of a specific gene product in a green alga. In opsin-deprived transformants, flash-induced photoreceptor currents (PC) are left unchanged. Moreover, photophobic responses as studied by motion analysis and phototaxis tested in a light-scattering assay were indistinguishable from the responses of untransformed wild-type cells. We conclude that phototaxis and photophobic responses in C. reinhardtii are triggered by an as yet unidentified rhodopsin species.

Sorption of radioactive contaminants by sediment from the Kara Sea.

The purpose of this study is to quantify some of the parameters needed to perform near-field modelling of sites in the Kara Sea that were impacted by the disposal of radioactive waste. The parameters of interest are: the distribution coefficients (Kd) for several important radionuclides, the mineralogy of the sediment, and the relationship of Kd to liquid-to-solid ratio. Sediment from the Kara Sea (location: 73 degrees 00'N, 58 degrees 00'E) was sampled from a depth of 287 m on August 23/24, 1992. Analysis of the material included mineralogy, grain size and total organic carbon (TOC). Uptake kinetics were determined for 85Sr, 137Cs, 241Am, 99Tc, 1251, 232U and 210Pb and distribution coefficients (Kd) were determined for these radionuclides using batch type experiments. Sorption isotherms, developed for 137Cs, 85Sr and 99Tc, were linear in each case. Increasing the liquid-to-solid ratio strongly increased uptake of 137Cs and moderately increased uptake of 99Tc. Analysis for anthropogenic radionuclides indicated the presence only of 239/240Pu in the sediment with the highest activity (at the top section of the core) being 0.420 Bq kg(-1). Other anthropogenic radionuclides were below detection limits.

A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii.

Although Chlamydomonas reinhardtii serves as the most popular algal model system, no efficient enzymatic selection marker for the nuclear transformation of wild-type cells is available. We sequenced an aminoglycoside 3'-phosphotransferase gene (aph) from Streptomyces rimosus. Though the derived protein sequence is homologous to members of APH type V, it constitutes a new type, named APHVIII. Since the aphVIII gene has a codon bias similar to that of the nuclear genome of green algae, the aphVIII coding sequence was fused to the 5'- and 3'-untranslated regions of the C. reinhardtii rbcS2 gene. C. reinhardtii transformants were capable of inactivating the antibiotics paromomycin, kanamycin, and neomycin, to which wild-type cells are sensitive. After addition of the 5'-region of hsp70A as a second promoter and insertion of the rbcS2 intron I, the transformation rate increased to two transformants per 1 x 10(5) cells, which is close to the efficiency of transforming auxotrophic strains with the homologous marker arg7. Transformation with the promoter-less aphVIII led to random gene fusion at high frequency. In an aphVIII-based reporter gene assay we have found a so far unknown promoter activity of the 3'-untranslated region of rbcS2, that may promote antisense RNA synthesis from the rbcS2 gene in vivo. We conclude that the aphVIII gene is a useful marker for nuclear transformation and promoter tagging of C. reinhardtii wild-type and probably other green algae.

Glucocorticoids inhibit lipopolysaccharide-induced up-regulation of arginase in rat alveolar macrophages.

1. As arginase by limiting nitric oxide (NO) synthesis may play a role in airway hyperresponsiveness and glucocorticoids are known to induce the expression of arginase I in hepatic cells, glucocorticoid effects on arginase in alveolar macrophages (AM Phi) were studied. 2. Rat AM Phi were cultured in absence or presence of test substances. Thereafter, nitrite accumulation, arginase activity, and the expression pattern of inducible NO synthase, arginase I and II mRNA (RT - PCR) and proteins (immunoblotting) were determined. 3. Lipopolyssacharides (LPS, 20 h) caused an about 2 fold increase in arginase activity, whereas interferon-gamma (IFN-gamma), like LPS a strong inducer of NO synthesis, had no effect. 4. Dexamethasone decreased arginase activity by about 25% and prevented the LPS-induced increase. Mifepristone (RU-486) as partial glucocorticoid receptor agonist inhibited LPS-induced increase by 45% and antagonized the inhibitory effect of dexamethasone. 5. Two different inhibitors of the NF-kappa B-pathway also prevented LPS-induced increase in arginase activity. 6. Rat AM Phi expressed mRNA and protein of arginase I and II, but arginase I expression was stronger. Arginase I mRNA and protein was not affected by IFN-gamma, but increased by LPS and this effect was prevented by dexamethasone. Both, LPS and IFN-gamma enhanced the levels of arginase II mRNA and protein, effects also inhibited by dexamethasone. As IFN-gamma did not affect total arginase activity, arginase II may represent only a minor fraction of total arginase activity. 7. In rat AM Phi glucocorticoids inhibit LPS-induced up-regulation of arginase activity, an effect which may contribute to the beneficial effects of glucocorticoids in the treatment of inflammatory airway diseases.

Nuclear factor-kappaB mediates simultaneous induction of inducible nitric-oxide synthase and Up-regulation of the cationic amino acid transporter CAT-2B in rat alveolar macrophages.

The connection between the regulation of L-arginine transport and nitric oxide (NO) synthesis was studied in rat alveolar macrophages. Lipopolysaccharides (LPSs) and interferon-gamma stimulated in the same concentration- and time-dependent manner NO synthesis (measured by nitrite accumulation) and L-[(3)H]arginine uptake. This correlated with an increased mRNA expression for iNOS and the cationic amino acid transporter CAT-2B (analyzed by reverse transcription-polymerase chain reaction), with the same kinetics observed for the up-regulation of both mRNAs. Because nuclear factor-kappaB (NF-kappaB) is essential for induction of iNOS its role for the regulation of CAT-2B expression and L-arginine transport was investigated. The NF-kappaB inhibitors pyrrolidine dithiocarbamate and N(alpha)-p-tosyl-L-lysine chloromethyl ketone abrogated LPS- and interferon-gamma-induced increase of nitrite accumulation and L-[(3)H]arginine uptake as well as up-regulation of iNOS and CAT-2B mRNA. LPS-induced increase in iNOS and CAT-2B mRNA was also suppressed by specific NF-kappaB decoy oligodesoxynucleotides, confirming the essential role of NF-kappaB for iNOS and CAT-2B expression. Dexamethasone did not affect the initial (5 h) LPS-induced increase of iNOS and CAT-2B mRNA, but down-regulated both mRNAs after prolonged (20 h) exposure and this was accompanied by partial inhibition of LPS-stimulated nitrite accumulation and L-[(3)H]arginine uptake. These findings demonstrate parallel regulation of the expression of iNOS and CAT-2B, and of NO synthesis and L-arginine uptake in rat alveolar macrophages. NF-kappaB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2B. The simultaneous up-regulation of CAT-2B with iNOS is considered as a mechanism to ensure a high substrate supply for iNOS.

MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro.

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.

A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii.

The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.

Biotin production under limiting growth conditions by Agrobacterium/Rhizobium HK4 transformed with a modified Escherichia coli bio operon.

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.

Purification, characterization, DNA sequence and cloning of a pimeloyl-CoA synthetase from Pseudomonas mendocina 35.

A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of approximately 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of approximately 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, pimelic acid-utilizing A) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.

Identification and function of cyclic nucleotide phosphodiesterase isoenzymes in airway epithelial cells.

Epithelial cells actively participate in inflammatory airway disease by liberating mediators such as arachidonate metabolites and cytokines. Inhibition of phosphodiesterases (PDEs) may be a useful anti-inflammatory approach. The PDE isoenzyme pattern and the effects of PDE inhibition on mediator generation were analyzed in primary cultures of human and porcine airway epithelial cells (AEC) and in the bronchial epithelial cell line BEAS-2B. PDE4 and PDE5 were detected in lysates of all cell types studied. In primary cultures of human AEC, the PDE4 variants PDE4A5, PDE4C1, PDE4D2, and PDE4D3 were identified by polymerase chain reaction analysis. Evidence of the recently described PDE7 was obtained by rolipram- insensitive cyclic adenosine monophosphate (cAMP) degradation, and its presence was verified by the demonstration of PDE7 messenger RNA. Primary cultures of human airway epithelium also expressed PDE1. Enhanced epithelial cAMP levels, induced by forskolin and PDE4 inhibition, increased formation of prostaglandin E2 (PGE2), but not of interleukin (IL)-8 or 15-hydroxyeicosatetraenoic acid (15-HETE) in airway epithelial cells. Increased cyclic guanosine monophosphate levels in these cells provoked by sodium nitroprusside and the PDE5 inhibitor zaprinast reduced the PGE2 synthesis, whereas 15-HETE and IL-8 formation were unchanged. The data suggest that PDE isoenzymes are important in airway inflammation and that PDE inhibitors exert anti-inflammatory effects by acting on AEC.