Increased Eicosanoid Levels in the Sugen/Chronic Hypoxia Model of Severe Pulmonary Hypertension

Inflammation and altered immunity are recognized components of severe pulmonary arterial hypertension in human patients and in animal models of PAH. While eicosanoid metabolites of cyclooxygenase and lipoxygenase pathways have been identified in the lungs from pulmonary hypertensive animals their role in the pathogenesis of severe angioobliterative PAH has not been examined. Here we investigated whether a cyclooxygenase-2 (COX-2) inhibitor or diethylcarbamazine (DEC), that is known for its 5-lipoxygenase inhibiting and antioxidant actions, modify the development of PAH in the Sugen 5416/hypoxia (SuHx) rat model. The COX-2 inhibitor SC-58125 had little effect on the right ventricular pressure and did not prevent the development of pulmonary angioobliteration. In contrast, DEC blunted the muscularization of pulmonary arterioles and reduced the number of fully obliterated lung vessels. DEC treatment of SuHx rats, after the lung vascular disease had been established, reduced the degree of PAH, the number of obliterated arterioles and the degree of perivascular inflammation. We conclude that the non-specific anti-inflammatory drug DEC affects developing PAH and is partially effective once angioobliterative PAH has been established.     

Antimicrobial peptides from the skins of North American frogs

North America is home to anuran species belonging to the families Bufonidae, Eleutherodactylidae, Hylidae, Leiopelmatidae, Ranidae, and Scaphiopodidae but antimicrobial peptides have been identified only in skin secretions and/or skin extracts of frogs belonging to the Leiopelmatidae (“tailed frogs”) and Ranidae (“true frogs”). Eight structurally-related cationic α-helical peptides with broad-spectrum antibacterial activity, termed ascaphins, have been isolated from specimens of Ascaphus truei (Leiopelmatidae) occupying a coastal range. Characterization of orthologous antimicrobial peptides from Ascaphus specimens occupying an inland range supports the proposal that this population should be regarded as a separate species A. montanus. Ascaphin-8 shows potential for development into a therapeutically valuable anti-infective agent. Peptides belonging to the brevinin-1, esculentin-1, esculentin-2, palustrin-1, palustrin-2, ranacyclin, ranatuerin-1, ranatuerin-2, and temporin families have been isolated from North American ranids. It is proposed that “ranalexins” represent brevinin-1 peptides that have undergone a four amino acid residue internal deletion. Current taxonomic recommendations divide North American frogs from the family Ranidae into two genera: Lithobates and Rana. Cladistic analysis based upon the amino acid sequences of the brevinin-1 peptides provides strong support for this assignment.     

Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs), such as Discs-Large (DLG), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between DLG and another PDZ scaffolding protein, SCRIBBLE (SCRIB), during the establishment of cell polarity in developing epithelia. A possible interaction between DLG and SCRIB at synaptic junctions has not yet been addressed. Likewise, the biochemical nature of this interaction remains elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. RESULTS: Here we report the isolation of a new DLG-interacting protein, GUK-holder, that interacts with the GUK domain of DLG and which is dynamically expressed during synaptic bouton budding. We also show that at Drosophila synapses DLG colocalizes with SCRIB and that this colocalization is likely to be mediated by direct interactions between GUKH and the PDZ2 domain of SCRIB. We show that DLG, GUKH, and SCRIB form a tripartite complex at synapses, in which DLG and GUKH are required for the proper synaptic localization of SCRIB. CONCLUSIONS: Our results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse.     

Sources of nitrate in rivers draining sixteen watersheds in the northeastern U.S.: Isotopic constraints

The feasibility of using nitrogen and oxygenisotope ratios of nitrate (NO₃ ^–) forelucidating sources and transformations ofriverine nitrate was evaluated in a comparativestudy of 16 watersheds in the northeastern U.S.A. Stream water was sampled repeatedly at theoutlets of the watersheds between January andDecember 1999 for determining concentrations,¹⁵N values, and ¹⁸Ovalues of riverine nitrate.In conjunction with information about land useand nitrogen fluxes,¹⁵N_nitrate and¹⁸O_nitrate values providedmainly information about sources of riverinenitrate. In predominantly forested watersheds,riverine nitrate had mean concentrations ofless than 0.4 mg NO₃ ^–-N L^–1,¹⁵N_nitrate values of lessthan +5, and ¹⁸O_nitratevalues between +12 and +19. This indicatesthat riverine nitrate was almost exclusivelyderived from soil nitrification processes withpotentially minor nitrate contributions fromatmospheric deposition in some catchments. Inwatersheds with significant agricultural andurban land use, concentrations of riverinenitrate were as high as 2.6 mg NO₃ ^–-NL^–1 with ¹⁵N_nitratevalues between +5 and +8 and¹⁸O_nitrate values generallybelow +15. Correlations between nitrateconcentrations, ¹⁵N_nitratevalues, and N fluxes suggest that nitrate inwaste water constituted a major, and nitrate inmanure a minor additional source of riverinenitrate. Atmospheric nitrate deposition ornitrate-containing fertilizers were not asignificant source of riverine nitrate inwatersheds with significant agricultural andurban land use. Although complementary studiesindicate that in-stream denitrification wassignificant in all rivers, the isotopiccomposition of riverine nitrate sampled at theoutlet of the 16 watersheds did not provideevidence for denitrification in the form ofelevated ¹⁵N_nitrate and¹⁸O_nitrate values. Relativelylow isotopic enrichment factors for nitrogenand oxygen during in-stream denitrification andcontinuous admixture of nitrate from theabove-described sources are thought to beresponsible for this finding.     

Controlled reduction of the humidity induces a shortcut recovery reaction in the photocycle of photoactive yellow protein

The photocycle of the blue-light photoreceptor protein Photoactive Yellow Protein (PYP) was studied at reduced relative humidity (RH). Photocycle kinetics and spectra were measured in thin films of PYP in which the relative humidity was set at values between 29 and 98% RH with saturated solutions of various salts. We show that in this range, approximately 200 water molecules per PYP molecule are released from the film. As humidity decreased, photocycle transition rates changed, until at low humidity (RH < 50%) an authentic photocycle was no longer observed and the absorption spectrum of the dark, equilibrium state of PYP started to shift to 355 nm, that is, to a form resembling that of pB(dark). At moderately reduced humidity (i.e., >50% RH), an authentic photocycle is still observed, although its characteristics differ from those in solution. As humidity decreases, the rate of ground state recovery increases, while the rate of depletion of the first red-shifted intermediate pR dramatically decreases. The latter observation contrasts all so-far known modulations of the rate of the transition of the red-shifted- to the blue-shifted intermediates of PYP, which is consistently accelerated by all other modulations of the mesoscopic context of the protein. Under these same conditions, the long-lived, blue-shifted intermediate was formed not only with slower kinetics than in solution but also to a smaller extent. Global analysis of these data indicates that in this low humidity environment the photocycle can take a different route than in solution, that is, part of pG recovers directly from pR. These experiments on wild-type PYP, in combination with observations on a variant of PYP obtained by site-directed mutagenesis (the E46Q mutant protein), further document the context dependence of the photocycle transitions of PYP and are relevant for the interpretation of results obtained in both spectroscopic and diffraction studies with crystalline PYP.     

Seedling establishment,underground kinetics,and clonal reiteration: How do Potentilla inclinata and Inula ensifolia get their multifunctional subterranean systems?

The development of a seedling into an adult plant comprises various underground processes. Time-lapse photography (TLP) makes them visible. This is documented for Potentilla inclinata (Rosaceae) and Inula ensifolia (Asteraceae).After germination, P. inclinata develops a taproot system. Contraction phenomena pull the basal part of the shoot at least 10 mm into the soil. Later, several adventitious roots are generated, and thus the root system changes to a fibrous one. This is followed by cloning without separation of the ramets.Seedlings of I. ensifolia develop a weak primary root. At an early stage, adventitious roots are formed at the leaf rosette. This fibrous root system exerts a strong pulling effect on the shoot. After one vegetation period the basis of the rosette is approx. 30 mm under the soil surface. Cloning includes the formation of many new horizontal shoots, which conquer new sites.These two examples show three functional steps common in the developmental progress of subterranean systems: (I) establishment of the seedling, (II) innovation and survival of the young plant, and (III) reiteration (cloning and dispersal). However, to accomplish these basic development steps the diversity of subterranean systems is enhanced by different organographical components.Furthermore, the development of subterranean systems is a dynamic process consisting of two kinetic processes: the vertical movement during seedling establishment, which brings the innovation buds to a safe soil position, and the horizontal movement during dispersal, which conquers new sites.     

Antibacterial Flavonoids and Other Compounds from the Aerial Parts of Vernonia guineensis Benth. (Asteraceae)

An extensive phytochemical study of the aerial parts of Vernonia guineensis Benth. (Asteraceae) led to the isolation of a new flavone, vernoguinoflavone and a naturally isolated glycerol ester, eicosanoic acid 2‐hydroxy‐1,3‐propanediyl ester, together with eighteen known secondary metabolites including quercetin, luteolin, vernopicrin, vernomelitensin, β‐amyrin, oleanolic acid, ursolic acid, lupeol, betulinic acid, β‐carotene, a mixture of stigmasterol and β‐sitosterol, β‐sitosterol‐3‐O‐β‐D‐glucoside, 2,3‐dihydroxypropyl heptacosanoate, pentacosanoic acid, docosan‐1‐ol, tritriacontan‐1‐ol, and heptatriacontan‐1‐ol. Eleven compounds are reported herein for the first time from this species. The structures of these compounds were elucidated on the basis of extensive spectroscopic analyses, particularly 1D and 2D NMR, and HR‐ESI‐MS and by comparison of their data with those reported in the literature. The crude extract, fractions and some isolated compounds were evaluated for their antibacterial activity against Gram‐negative bacteria: Escherichia coli (ATCC 25922), Shigella flexineri (NR 518), Salmonella muenchen, Salmonella typhimurium and Salmonella typhi (ATCC 19430). All the tested compounds demonstrated inhibitory activities against the tested enteric bacteria with MIC values ranging from 3.12 to 100 μg/ml. Three flavonoids isolated from the most active fraction demonstrated the best bioactivities against Escherichia coli, Salmonella muenchen and Salmonella typhimurium with MIC values ranging from 3.12 to 25 μg/mL.     

Comparative titration of arginyl residues in purified D-β-hydroxybutyrate apodehydrogenase and in the reconstituted phospholipid-enzyme complex

In order to titrate and understand the role of arginyl residues of D-β-hydroxybutyrate dehydrogenase, arginyl specific reagents: butanedione, 1,2-cyclohexanedione and phenylglyoxal were incubated with three different forms of the enzyme; native enzyme (inner mitochondrial membrane bound), purified apoenzyme (phospholipid -free) and phospholipid-enzyme complex (reconstituted active form).After complete inactivation of the enzyme by [¹⁴C]-phenylglyoxal, the number of modified arginyl residues was different: one with the lipid-free apoenzyme and three with the phospholipid-enzyme complex, suggesting a conformational change of the enzyme triggered by the presence of phospholipids.After exhaustive chemical modification either of the apoenzyme or of the phospholipid-enzyme complex with [¹⁴C]-phenylglyoxal, four arginyl residues were titrated indicating that these residues are located in the hydrophilic part of the enzyme, not interacting with phospholipids.Reconstituted enzyme inactivated by butanedione could no longer bind a pseudosubstrate (succinate) which indicates that an arginyl residue is involved in the enzyme-substrate complex formation.The values of second order rate constants of D-β-hydroxybutyrate dehydrogenase inactivation by butanedione and 1,2-cyclohexanedione were unchanged with the three enzyme forms, suggesting that phospholipids are not involved in the substrate binding mechanism.