Innate defense against influenza A virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein D

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.     

GABAB antagonists: Enantiopharmacology of 5-amino-4-hydroxy-2-methylvaleric acid and X-ray structure of the eutomer

We have previously shown that (R)-5-amino-4-hydroxyvaleric acid [(R)-4-OH-DAVA] and (S)-2-OH-DAVA bind to GABA_B receptor sites and antagonize GABA_B receptor-mediated function in a stereoselective manner. Furthermore, we have identified energy-minimized superimposable conformations of (R)-4-OH- and (S)-2-OH-DAVA which are assumed to reflect the receptor-active conformations of these compounds. This paper describes the in vitro enantiopharmacology of 5-amino-4-hydroxy-2-methylvaleric acid (2-Me-4-OH-DAVA). Whereas none of the four stereoisomers showed significant affinity for GABA_A receptor sites or GABA uptake mechanisms in rat brain synaptic membranes, (2R,4R)-2-Me-4-OH-DAVA was shown to inhibit stereoselectively the binding of [³H]GABA to rat brain GABA_B receptor sites (IC₅₀ = 14 ± 4 μM). (2R,4R)-2-Me-4-OH-DAVA (K_i = 36 μM) and, with much lower potency, (2S,4R)-2-Me-4-OH-DAVA (K_i = 370 μM) stereoselectively antagonized GABA_B receptor-mediated function in the isolated guinea pig ileum. The structure of the eutomer, (2R,4R)-2-Me-4-OH-DAVA, was established by an X-ray crystallographic analysis, and the solid-state conformation of (2R,4R)-2-Me-4-OH-DAVA was compared with the proposed receptor-active conformations of (R)-4-OH-DAVA and (S)-2-OH-DAVA. © 1995 Wiley-Liss, Inc.     

Blood vessels and the occurrence of arteriovenous anastomoses in cephalic heat loss areas of mallards,Anas platyrhynchos (Aves)

Summary 1. The blood supply to cephalic heat loss areas (nasal and oropharyngeal mucosa, bill, eyelids) was studied in mallards by using plastic corrosion casts. The structure and organization of the blood vessels, as well as the occurrence of arteriovenous anastomoses (AVAs), were examined by scanning electron microscopy of vascular casts and by paraffin sections.2. Submucosal venous plexuses (cavernous tissue) are present in the nasal cavity, tongue, and lateral margins of the palate. These plexuses receive blood from post-capillary venules, but may also receive a non-nutritive component via numerous AVAs.3. High densities of AVAs were found in the eyelids and in the tip of the bill. In the tongue and nasal mucosa, the AVAs decreased in number caudally. The reason for regional differences in the density of AVAs is discussed in relation to variation in mechanical and thermal stimulation of the tissues.4. The connection of the different heat loss areas with the Rete ophthalmicum, which is a countercurrent heat exchanger important for brain cooling, is pointed out. The vascular pattern of the head suggests that sphincteric veins are involved in regulating the venous return from the evaporative surfaces of the nasal cavity and palate. One of these veins had, in addition to the normal circular smooth muscle fibres, a conspicuous component of longitudinally arranged, subendothelial, smooth muscle fibres.     

Rad52 and Rad59 exhibit both overlapping and distinct functions

Homologous recombination is an important pathway for the repair of DNA double-strand breaks (DSBs). In the yeast Saccharomyces cerevisiae, Rad52 is a central recombination protein, whereas its paralogue, Rad59, plays a more subtle role in homologous recombination. Both proteins can mediate annealing of complementary single-stranded DNA in vitro, but only Rad52 interacts with replication protein A and the Rad51 recombinase. We have studied the functional overlap between Rad52 and Rad59 in living cells using chimeras of the two proteins and site-directed mutagenesis. We find that Rad52 and Rad59 have both overlapping as well as separate functions in DSB repair. Importantly, the N-terminus of Rad52 possesses functions not supplied by Rad59, which may account for its central role in homologous recombination.     

Identification and Characterization of a Chitin-binding Protein Purified from Coelomic Fluid of the Lugworm Arenicola marina Defining a Novel Protein Sequence Family

We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm A. marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass of 27 and 31 kDa in the reduced state. The N-terminal amino acid sequences were identical in these two bands. In the unreduced state, a complex band pattern was observed with bands from 35 kDa to more than 200 kDa. Two different full-length clones encoding polypeptides of 241 and 243 amino acids, respectively, were isolated from a coelomocyte cDNA library. The two clones, designated AML-1a and AML-1b, were 92% identical at the protein level and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a in the reduced state lacked a 27 kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed that AML-1 is expressed by coelomocytes in the nephridium and in round cells in the epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components.     

RNA chaperone activity of translation initiation factor IF1

Translation initiation factor IF1 is an indispensable protein for translation in prokaryotes. No clear function has been assigned to this factor so far. In this study we demonstrate an RNA chaperone activity of this protein both in vivo and in vitro. The chaperone assays are based on in vivo or in vitro splicing of the group I intron in the thymidylate synthase gene (td) from phage T4 and an in vitro RNA annealing assay. IF1 wild-type and mutant variants with single amino acid substitutions have been analyzed for RNA chaperone activity. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Furthermore, both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.     

A versatile selection system for folding competent proteins using genetic complementation in a eukaryotic host

Recombinant expression of native or modified eukaryotic proteins is pivotal for structural and functional studies and for industrial and pharmaceutical production of proteins. However, it is often impeded by the lack of proper folding. Here, we present a stringent and broadly applicable eukaryotic in vivo selection system for folded proteins. It is based on genetic complementation of the Schizosaccharomyces pombe growth marker gene invertase fused C‐terminally to a protein library. The fusion proteins are directed to the secretion system, utilizing the ability of the eukaryotic protein quality‐control systems to retain misfolded proteins in the ER and redirect them for cytosolic degradation, thereby only allowing folded proteins to reach the cell surface. Accordingly, the folding potential of the tested protein determines the ability of autotrophic colony growth. This system was successfully demonstrated using a complex insertion mutant library of TNF‐α, from which different folding competent mutant proteins were uncovered.     

A novel FRET pair for detection of parallel DNA triplexes by the LightCycler

Background  

Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic pH quenching of FAM is not very suitable, since the fluorescence of FAM is strongly pH dependent and drops with acidic pH.