Structures of the lectins from Pseudomonas aeruginosa: insight into the molecular basis for host glycan recognition

The opportunistic human pathogen Psuedomonas aeruginosa produces two lectins in close association with virulence factors: PA-IL adn PA-IIL, which bind to galactose- and fucose/mannose-containing glycoconjugates, respectively. We review here the structural aspects of these lectins relative to their putative roles in host recognition, cell surface adhesion and biofilm formation.     

Morphologic and genetic variability in the marine planktonic ciliate Laboea strobila Lohmann, 1908 (Ciliophora, Oligotrichia), with notes on its ontogenesis

Laboea strobila Lohmann, 1908 is a conspicuous oligotrich ciliate in the marine plankton. In order to compare different populations, the morphology of specimens from the Mediterranean Sea, North Sea, and Irish Sea was investigated using live observation, protargol impregnation, and scanning electron microscopy. Furthermore, the PCR-amplified products of the SSrRNA gene from a monoclonal culture of L. strobila from the Mediterranean Sea were sequenced and aligned with sequences of other oligotrichs, including a population of L. strobila from the Atlantic coast of the USA. Finally, the data from the ecological literature were summarized and the cultivation methods were described. The SSrRNA gene sequences of the two distantly located L. strobila populations from the North Atlantic are identical. Likewise, the morphometrics of most populations so far investigated after protargol impregnation (i.e. from the North Atlantic) do not show obvious differences. In all computed phylogenetic trees, L. strobila groups with Strombidium species, forming a monophyletic taxon corresponding to the subclass Oligotrichia. These results are corroborated by the ontogenetic comparison. Since no type species was fixed for Laboea Lohmann, 1908, L. strobila was designated in the present paper.     

dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency

Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro. We show that recombinant betaretroviral NC-dUTPase and dUTPase are both inefficient catalysts compared with all other dUTPases. Dynamic light scattering and gel filtration confirm that the homotrimeric organization, common among dUTPases, is retained in the NC-dUTPase fusion protein. The betaretroviral dUTPase has been crystallized and single crystals contain homotrimers. Oligonucleotide and Zn2+ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase. Binding of the hexanucleotide ACTGCC or the octanucleotide (TG)4 to NC-dUTPase modulates enzymatic function, indicating that the low catalytic activity may be compensated by adequate localization.     

Increased sensitivity to endothelial nitric oxide (NO) contributes to arterial normotension in mice with vascular smooth muscle-selective deletion of the atrial natriuretic peptide (ANP) receptor

Atrial natriuretic peptide (ANP) plays a key regulatory role in arterial blood pressure homeostasis. We recently generated mice with selective deletion of the ANP receptor, guanylyl cyclase-A (GC-A), in vascular smooth muscle (SMC GC-A knockout (KO) mice) and reported that resting arterial blood pressure was completely normal in spite of clear abolition of the direct vasodilating effects of ANP (Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M., Potthast, R., Zetsche, B., Hammer, R. E., Herz, J., and Kuhn M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7142-7147). The purpose of this study was to clarify mechanisms compensating for the missing vasodilator responses to ANP. In particular, we analyzed the effect of the endothelial, cGMP-mediated vasodilators C-type natriuretic peptide and nitric oxide (NO). In isolated arteries from SMC GC-A KO mice, the vasorelaxing sensitivity to sodium nitroprusside and the endothelium-dependent vasodilator, acetylcholine, was significantly greater than in control mice. There was no difference in responses to C-type natriuretic peptide or to the activator of cGMP-dependent protein kinase I, 8-para-chlorophenylthio-cGMP. The aortic expression of soluble GC (sGC), but not of endothelial NO synthase or cGMP-dependent protein kinase I, was significantly increased in SMC GC-A KO mice. Chronic oral treatment with the NO synthase inhibitor N(w)-nitro-l-arginine methyl ester increased arterial blood pressure, the effect being significantly enhanced in SMC GC-A KO mice. We conclude that SMC GC-A KO mice exhibit a higher vasodilating sensitivity to NO. This can be attributed to an enhanced expression of sGC, whereas the expression and/or activity levels of downstream cGMP-effector pathways are not involved. Increased vasodilating responsiveness to endothelial NO contributes to compensate for the missing vasodilating effect of ANP in SMC GC-A KO mice.     

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.     

Determination of 11-deoxycortisol (Reichstein's compound S) in human plasma by clinical isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection

A first assay based on stable isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS) has been developed for plasma 11-deoxycortisol (Reichstein's compound S), the leading hormonal marker of 11beta-hydroxylase deficiency. A suitable internal standard being unavailable, we synthesized dideuterated 11-deoxycortisol according to a newly devised synthetic procedure. 17,21-Dihydroxy-pregna-1,4-diene-3,20-dione underwent selective deuteration using Wilkinson's catalyst. Our product [1alpha,2alpha-2H2]11-deoxycortisol was obtained in good yield (35.6%) and high isotopic purity (0.1% 2H0, 99.9% 2H2). Structural confirmation was done by MS and NMR. Our plasma work up consisted of equilibration of plasma with internal standard ([1alpha,2alpha-2H2]11-deoxycortisol), solid phase extraction with Extrelut NT columns, a clean up step using Sephadex LH-20 mini columns and preparation of heptafluorobutyrates as derivatives. Quantification was achieved by selected ion monitoring of m/z 465.40 (analyte) and m/z 467.40 (internal standard). One hundred twenty picograms of 11-deoxycortisol gave a signal to noise ratio of 10. Calibration plot was linear. Spiking experiments showed good accuracy with relative errors <3.0%. Intraassay precision CV was 4.78% and interassay precision CV was 4.56%. We succeeded in integrating our new analyte into our already existing multisteroid ID/GC-MS plasma assay, which now, in its expanded version, is capable of determining all major diagnostic steroids of androgen related disorders in a single profile: 11-deoxycortisol, 17alpha-hydroxyprogesterone, testosterone, 4-androstenedione, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, androstanediol and 5alpha-dihydrotestosterone. The diagnostic potential of our multisteroid ID/GC-MS assay, the small amounts of plasma (0.5 ml) required, the rapid and convenient sample work up, the application of benchtop GC-MS instrumentation, and highest specificity offered by mass spectrometric detection prove our assay suitable for routine clinical use, especially in pediatric endocrinology.