Three-dimensional structure of horse liver alcohol dehydrogenase at 2-4 A resolution.

The crystal structure analysis of horse liver alcohol dehydrogenase has been extended to 2.4 Å resolution. From the corresponding electron density map of the apoenzyme we have determined the positions of the 374 amino acids in the polypeptide chain of each subunit.The coenzyme binding domain of the subunit comprises residues 176 to 318. 45% of these residues are helical and 32% are in the central six-stranded pleated sheet structure. The positions and orientations of the helices with respect to the pleated sheet indicate a possible folding mechanism for this part of the subunit structure. The coenzyme analogue ADP-ribose binds to this domain in a position and orientation very similar to coenzyme binding to lactate dehydrogenase. The adenine part binds in a hydrophobic pocket, the adenosine ribose is hydrogen-bonded to the side chain of Asp223, the pyrophosphate is positioned by interaction with Arg47 and the nicotinamide ribose is 6Å away from the catalytic zinc atom.The catalytic domain is mainly built up from three distinct antiparallel pleated-sheet regions. Residues within this domain provide ligands to the catalytic zinc atom; Cys46, His67 and Cys174. An approximate tetrahedral coordination of this zinc is completed by a water molecule or hydroxyl ion depending on the pH. Residues 95 to 113 form a lobe that binds the second zinc atom of the subunit. This zinc is liganded in a distorted tetrahedral arrangement by four sulphur atoms from the cysteine residues 97, 100, 103 and 111. The lobe forms one side of a significant cleft in the enzyme surface suggesting that this region might constitute a second catalytic centre of unknown function.The two domains of the subunit are separated by a crevice that contains a wide and deep hydrophobic pocket. The catalytic zinc atom is at the bottom of this pocket, with the zinc-bound water molecule projecting out into the pocket. This water molecule is hydrogen-bonded to the side chain of Ser48 which in turn is hydrogen-bonded to His51. The pocket which in all probability is the binding site for the substrate and the nicotinamide moiety of the coenzyme, is lined almost exclusively with hydrophobic side chains. Both subunits contribute residues to each of the two substrate binding pockets of the molecule. The only accessible polar groups in the vicinity of the catalytic centre are Ser48 and Thr178 apart from zinc and the zinc-bound water molecule.     

Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents

To fully understand the function of the Cu- and Zn-containing superoxide dismutases in normal and disordered cells, it is essential to study protein variants with full metal contents. We describe the use of an Escherichia coli-based expression system for the overproduction of human intracellular wild type CuZn-superoxide dismutase (SOD), the CuZnSOD variant F50E/G51E (monomeric), two amyotrophic lateral sclerosis-related mutant CuZnSOD variants (D90A and G93A), and PseudoEC-SOD, all with high Cu contents. This system is based on coexpression of the SOD variants with the yeast copper chaperone yCCS during growth in a medium supplemented with Cu(2+) and Zn(2+). The recombinant SOD enzymes were all found in the cytosol and represented 30-50% of the total bacterial protein. The enzymes were purified to homogeneity and active enzymes were obtained in high yield. The resulting proteins were characterized through immunochemical reactivity and specific activity analyses, in conjunction with mass-, photo-, and atomic absorption-spectroscopy.     

Bradykinin upregulates IL-8 production in human gingival fibroblasts stimulated by interleukin-1beta and tumor necrosis factor alpha

The proinflammatory mediator bradykinin (BK) is suggested to play an important role in the pathogenesis of various inflammatory diseases including periodontitis. In this study, BK per se stimulated interleukin-8 (IL-8) production in human gingival fibroblasts in vitro. Furthermore, BK upregulated the stimulatory effect of the cytokines IL-1beta and TNFalpha on the production of IL-8. The stimulatory effect of BK on the IL-1beta- or TNFalpha-stimulated IL-8 production was reduced in the presence of BK B2 receptor antagonist HOE 140, whereas the B1 receptor antagonist Lys-(des-arg9, Leu8)-BK had no effect. Similar to BK, the calcium ionophore A23187 also upregulated the stimulatory effect of IL-1beta and TNFalpha on IL-8 production. The protein kinase C (PKC) inhibitor bisindolylmaleimide, BIS, significantly reduced the stimulatory effect of BK on IL-1beta and TNFalpha increased IL-8 production but did not affect the production of IL-8 stimulated by cytokines alone. The specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 reduced IL-8 production stimulated by the combination of BK and IL-1beta as well as the IL-1beta-stimulated IL-8 production. In conclusion, this study shows that BK upregulates IL-1beta- and TNFalpha-stimulated IL-8 production via BK B2 receptor and that PKC signal pathway seems to be involved in the upregulation of the cytokine-induced IL-8 production in gingival fibroblasts. This stimulatory effect of BK on IL-8 production may contribute to the maintenance of the gingival inflammation and enhanced risk for destruction of gingival connective tissue.     

Tyrosine protonation changes in bacteriorhodopsin. A Fourier transform infrared study of BR548 and its primary photoproduct

The structural alterations which occur in bacteriorhodopsin (bR) during dark adaptation (BR570----BR548) and the primary phototransition of the dark photocycle (BR548----KD610) have been investigated by Fourier transform infrared and UV difference spectroscopy. Possible contributions of tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exists in BR570 at physiological temperature and that it protonates during formation of BR548. A tyrosinate protonation has also been observed at low temperature during the primary phototransition of BR570 to the red-shifted photoproduct K630 (1). However, we now find that no tyrosine protonation change occurs during the primary phototransition of BR548 to the red-shifted intermediate KD610. Through analysis of bR containing isotopically labeled retinals, it was also determined that the chromophore of KD610 exits in a 13-trans, 15-cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570, BR548, and K630, it appears that only the 13-trans,15-trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with the Schiff base positive charge in the BR570 chromophore. Isomerization of the chromophore about either the C13 = C14 or C = N bond disrupts this interaction causing a protonation of the tyrosinate.     

Über Vorkommen und Physiologie der Phosphatide in tierischen Zellen,besonders im Nervensystem.

Zusammenfassung In der vorliegenden Arbeit wurde chemisch und histologisch bewiesen, daß in den Ganglienzellen sowie in deren Ausläufern, den Dendriten und Achsenzylindern, ferner in den Gliazellen und schließlich in der Stäbchenstruktur der Nierenzellen Phosphatide enthalten sind, die einen dominierenden Bestandteil darstellen. In den Markscheiden fehlen die Phosphatide.Ferner werden einige gliöse Strukturen, besonders von marginalem und perivaskulärem Typ mit den zugehörigen Membranae limitantes beschrieben. Auch wurde das Vorhandensein von Verbindungen zwischen den Astrocyten und der Oligodendroglia festgestellt.In der Untersuchung wird ein Versuch vorgelegt, die festgestellten histologischen und chemischen Verhältnisse physiologisch zu erklären.Es ist für mich eine besonders angenehme Pflicht, Herrn Professor Dr. John Runnström, Stockholm, für seine liebenswürdige Unterstützung meiner Untersuchung mit Materialien und seine Durchmusterung meiner mikroskopischen Präparationen meinen besten Dank aussprechen. Ebenso möchte ich Herrn Dr. L. Brundin, Stockholm, für seinen bereitwilligen Beistand bei der Anfertigung der Zeichnungen danken.     

Aberrant association between vascular endothelial growth factor receptor-2 and VE-cadherin in response to vascular endothelial growth factor-a in Shb-deficient lung endothelial cells

Vascular permeability is a hallmark response to the main angiogenic factor VEGF-A and we have previously described a reduction of this response in Shb knockout mice. To characterize the molecular mechanisms responsible for this effect, endothelial cells were isolated from lungs and analyzed in vitro. Shb deficient endothelial cells exhibited less migration in a scratch wound-healing assay both under basal conditions and after vascular endothelial growth factor-A (VEGF-A) stimulation, suggesting a functional impairment of these cells in vitro. Staining for VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR-2) showed co-localization in adherens junctions and in intracellular sites such as the perinuclear region in wild-type and Shb knockout cells. VEGF-A decreased the VE-cadherin/VEGFR-2 co-localization in membrane structures resembling adherens junctions in wild-type cells whereas no such response was noted in the Shb knockout cells. VE-cadherin/VEGFR-2 co-localization was also recorded using spinning-disk confocal microscopy and VEGF-A caused a reduced association in the wild-type cells whereas the opposite pattern was observed in the Shb knockout cells. The latter expressed slightly more of cell surface VEGFR-2. VEGF-A stimulated extracellular-signal regulated kinase, Akt and Rac1 activities in the wild-type cells whereas no such responses were noted in the knockout cells. We conclude that aberrant signaling characteristics with respect to ERK, Akt and Rac1 are likely explanations for the observed altered pattern of VE-cadherin/VEGFR-2 association. The latter is important for understanding the reduced in vivo vascular permeability response in Shb knockout mice, a phenomenon that has patho-physiological relevance.     

Comparative proteomic profiles of the marine cyanobacterium Trichodesmium erythraeum IMS101 under different nitrogen regimes

Trichodesmium is a marine filamentous diazotrophic cyanobacterium and an important contributor of "new" nitrogen in the oligotrophic surface waters of the tropical and sub-tropical oceans. It is unique in that it exclusively fixes N(2) at daytime, although it belongs to the non-heterocystous filamentous segment of the cyanobacterial radiation. Here we present the first quantitative proteomic analysis of Trichodesmium erythraeum IMS101 when grown under different nitrogen regimes using 2-DE/MALDI-TOF-MS. Addition of combined nitrogen (NO3-) prevented development of the morphological characteristics of the N(2)-fixing cell type (diazocytes), inhibited expression of the nitrogenase enzyme subunits and consequently N(2) fixation activity. The diazotrophic regime (N(2) versus NO3- cultures) elicited the differential expression of more than 100 proteins, which represented 13.5% of the separated proteins. Besides proteins directly related to N(2) fixation, proteins involved in the synthesis of reducing equivalents and the generation of a micro-oxic environment were strongly up-regulated, as was in particular Dps, a protein related to iron acquisition and potentially other vital cellular processes. In contrast, proteins involved in the S-adenosylmethionine (SAM) cycle, synthesis of amino acids and production of carbon skeletons for storage and synthesis of amino acids were suppressed. The data are discussed in the context of Trichodesmium's unusual N(2)-fixing physiology.