The intimate and mysterious relationship between proton channels and NADPH oxidase

Voltage gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Although these molecules are distinct entities, with no proven physical interaction, their presence and activity in many cells appears to be coordinated. We describe these interactions and discuss several types of mechanisms that might explain them.     

Topology is the Principal Determinant in the Folding of a Complex All-alpha Greek Key Death Domain from Human FADD

In order to elucidate the relative importance of secondary structure and topology in determining folding mechanism, we have carried out a phi-value analysis of the death domain (DD) from human FADD. FADD DD is a 100 amino acid domain consisting of six anti-parallel alpha helices arranged in a Greek key structure. We asked how does the folding of this domain compare with that of (a) other all-alpha-helical proteins and (b) other Greek key proteins? Is the folding pathway determined mainly by secondary structure or is topology the principal determinant? Our Φ-value analysis reveals a striking resemblance to the all-beta Greek key immunoglobulin-like domains. Both fold via diffuse transition states and, importantly, long-range interactions between the four central elements of secondary structure are established in the transition state. The elements of secondary structure that are less tightly associated with the central core are less well packed in both cases. Topology appears to be the dominant factor in determining the pathway of folding in all Greek key domains.     

Proton transport coupled ATP synthesis by the purified yeast H-ATP synthase in proteoliposomes

The H⁺/ATP synthase from yeast mitochondria, MF₀F₁, was purified and reconstituted into liposomes prepared from phosphatidylcholine and phosphatidic acid. Analysis by mass spectrometry revealed the presence of all subunits of the yeast enzyme with the exception of the K-subunit. The MF₀F₁ liposomes were energized by acid-base transitions (ΔpH) and a K⁺/valinomycin diffusion potential (Δφ). ATP synthesis was completely abolished by the addition of uncouplers as well as by the inhibitor oligomycin. The rate of ATP synthesis was optimized as a function of various parameters and reached a maximum value (turnover number) of 120 s^{− 1} at a transmembrane pH difference of 3.2 units (at pH_in = 4.8 and pH_out = 8.0) and a Δφ of 133 mV (Nernst potential). Functional studies showed that the monomeric MF₀F₁ was fully active in ATP synthesis. The turnover increased in a sigmoidal way with increasing internal and decreasing external proton concentration. The dependence of the turnover on the phosphate concentration and the dependence of K_M on pH_out indicated that the substrate for ATP synthesis is the monoanionic phosphate species H₂PO₄^−.     

Curcumin dramatically enhances retinoic acid-induced superoxide generating activity via accumulation of p47-phox and p67-phox proteins in U937 cells

The membrane bound cytochrome b558 composed of large gp91-phox and small p22-phox subunits, and cytosolic proteins p40-, p47- and p67-phox are important components of superoxide ()-generating system in phagocytes and B lymphocytes. A lack of this system in phagocytes is known to cause serious life-threatening infections. Here, we describe that curcumin, a polyphenol responsible for the yellow color of curry spice turmeric, dramatically activates the -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and curcumin, the -generating activity increased more than 4-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and curcumin slightly enhanced gene expressions of the five components compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and curcumin caused remarkable accumulation of protein levels of p47-phox (to 7-fold) and p67-phox (to 4-fold) compared with those of the RA-treatment alone. These results suggested that curcumin dramatically enhances RA-induced -generating activity via accumulation of cytosolic p47-phox and p67-phox proteins in U937 cells. Therefore, it should have the potential as an effective modifier in therapy of leukemia and/or as an immunopotentiator.     

Molecular characterization of β1,4-galactosyltransferase 7 genetic mutations linked to the progeroid form of Ehlers-Danlos syndrome (EDS)

β1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme initiating glycosaminoglycan (GAG) synthesis. Based on in vitro and ex vivo kinetics studies and structure-based modelling, we molecularly characterized β4GalT7 mutants linked to the progeroid form of Ehlers-Danlos syndrome (EDS), a severe connective tissue disorder. Our results revealed that loss of activity upon L206P substitution due to altered protein folding is the primary cause for the GAG synthesis defect in patients carrying the compound A186D and L206P mutations. We showed that R270C substitution strongly reduced β4GalT7 affinity towards xyloside acceptor, thus affecting GAG chains formation. This study establishes the molecular basis for β4GalT7 defects associated with altered GAG synthesis in EDS.     

Modeling species-specific diacylglycerol dynamics in the RAW 264.7 macrophage

A mathematical model of the G protein signaling pathway in RAW 264.7 macrophages downstream of P2Y₆ receptors activated by the ubiquitous signaling nucleotide uridine 5’-diphosphate is developed. The model, which is based on time-course measurements of inositol trisphosphate, cytosolic calcium, and diacylglycerol, focuses particularly on differential dynamics of multiple chemical species of diacylglycerol. When using the canonical pathway representation, the model predicted that key interactions were missing from the current network structure. Indeed, the model suggested that accurate depiction of experimental observations required an additional branch to the signaling pathway. An intracellular pool of diacylglycerol is immediately phosphorylated upon stimulation of an extracellular receptor for uridine 5’-diphosphate and subsequently used to aid replenishment of phosphatidylinositol. As a result of sensitivity analysis of the model parameters, key predictions can be made regarding which of these parameters are the most sensitive to perturbations and are therefore most responsible for output uncertainty.     

Distribution of MAP1A,MAP1B,and MAP2A&;B during layer formation in the optic tectum of developing chick embryos

The expression patterns of three microtubule-associated proteins (MAP1A, MAP1B, and MAP2A&B) were investigated in the developing optic tectum. Expression of MAP1B and middle-molecular-weight peptide of neurofilament (NF-M) was first observed in the same mesencephalic cells on day 3 of incubation, indicating that neuroblasts had been produced. At day 5, MAP1A and MAP2A&B expression appeared in the cellular layer containing the first neuroblasts that differentiate into large multipolar cells. The NF-M⁺ neurites in the striatum album centrale (SAC) and the striatum opticum (SO) were MAP1B⁺ up to day 19, but the intensity of MAP1B immunoreactivity decreased with development. All three MAPs were expressed in large multipolar neurons in the developing stratum griseum centrale from the beginning of maturation. Stratum griseum et fibrosum centrale cellular layers, containing radially arranged piriform neurons, were MAP1A^–/MAP2A&B^– on day 11 but became MAP1A⁺/MAP2A&B⁺ during later stages. These results suggest that the timing of MAP expression in neuronal maturation of large multipolar cells differs from that of piriform cells. The expression of MAPs has revealed specific cellular events in the developing optic tectum. Based on our observations, the development of the optic tectum can be divided into four periods.     

Regeneration of fertile plants from Helianthus nuttallii T&G and Helianthus giganteus L. mesophyll protoplasts

Interspecific hybridisation in the genus Helianthus via somatic cell fusion is thought to play an important role in future sunflower breeding programs. The establishment of this technique requires, however, the development of single-cell-regeneration protocols. For this purpose, we applied a regeneration protocol recently developed for Helianthus annuus L. to mesophyll protoplasts of two wild sunflowers (H. nuttallii T&G, H. giganteus L). Protoplasts of both species were embedded in agarose droplets and covered by liquid mKM medium. After 4–5 weeks, callus was transferred onto solid differentiation medium yielding plating efficencies of 1.5% (H. nuttallii) and 2.5% (H. giganteus). Emerging shoots were elongated on hormone-free medium, and root formation was induced by an NAA treatment. Regenerated plants were transferred to the greenhouse where they grew up to a height of 2 m and flowered after 3 months. Seeds were harvested from regenerated plants of both species. Received: 22 October 1996 / Revision received: 30 December 1996 / Accepted: 30 January 1997     

Phenotypic modulation of cultured endothelial cells in collagen matrices induced by tumor necrosis factor alpha

Summary The effect of tumor necrosis factor alpha on vascular endothelial cells was analyzed using a collagen-embedded, three-dimensional culture system, focusing on angiogenesis and expression of cell adhesion molecules. When the endothelial cells were cultured between two layers of type-I collagen gel, they reorganized into a network of branching and anastomosing tubular structures. Once the structure was formed, the cells did not undergo further division. Addition of tumor necrosis factor alpha at 10 to 500 U/ml to the overlaid culture medium inhibited this tube-forming process and enhanced their survival, whereas it suppressed cell growth in monolayer. To test its effect on the expression of cell adhesion molecules, the collagen was digested, and the dispersed cells were stained with anti-intercellular adhesion molecule-1 and endothelial-leukocyte adhesion molecule-1 monoclonal antibodies. Tumor necrosis factor alpha upregulated the expressions of both molecules for an extended period of time. Even in the absence of tumor necrosis factor alpha, the cells embedded in collagen matrices expressed small amounts of these adhesion molecules. These results indicate that endothelial cells display phenotypic changes in collagen matrices and modulatory response to tumor necrosis factor alpha.     

Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction

Restriction endonuclease cleavage maps of five DNA fragments carrying genes for neomycin phosphotransferase and neomycin acetyltransferase (from Streptomyces fradiae), viomycin phosphotransferase (from S. vinaceus), and ribosomal methylases determining resistance to thiostrepton (from S. azureus) and MLS antibiotics (from S. erythreus) are described, together with a map for the SLP1.2 Streptomyces plasmid used to isolate the fragments. Construction of a versatile Streptomyces cloning vector (pIJ61) is reported. pIJ61 carries neomycin phosphotransferase and thiostrepton resistance genes and has unique BamHI and PstI sites which will allow clone recognition by insertional inactivation of neomycin resistance; cloning sites for several other endonucleases are also present. pIJ28, a shuttle vector for Streptomyces and E. coli, carries neomycin resistance and the SLP1.2 and pBR322 replicons.